Methods and compositions for treating and diagnosing diseases

ABSTRACT

Methods and compositions for diagnosing and treating diseases, particularly cancer, associated with differential expression of cancer-associated targets (CAT) in disease cells compared to healthy cells are provided. Also provided are antagonists and agonists of CAT, and methods for screening agents that modulate CAT level or activity in vivo or in vitro.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. non-provisionalapplication Ser. No. 13/670,293, filed Nov. 6, 2012, which is adivisional application of U.S. non-provisional application Ser. No.13/339,833, filed Dec. 29, 2011 (issued as U.S. Pat. No. 8,334,106 onDec. 18, 2012), which is a divisional application of U.S.non-provisional application Ser. No. 12/504,826, filed Jul. 17, 2009(issued as U.S. Pat. No. 8,110,313 on Feb. 7, 2012), which is adivisional application of U.S. non-provisional application Ser. No.11/635,029, filed Dec. 7, 2006 (issued as U.S. Pat. No. 7,582,441 onSep. 1, 2009), which is a continuation application of U.S.non-provisional application Ser. No. 11/581,732, filed Oct. 17, 2006,which claims priority to U.S. provisional application Ser. No.60/819,615, filed on Jul. 11, 2006, and to U.S. provisional applicationSer. No. 60/819,614, filed on Jul. 11, 2006, and to U.S. provisionalapplication Ser. No. 60/819,613, filed on Jul. 11, 2006, and to U.S.provisional application Ser. No. 60/818,503, filed on Jul. 6, 2006, andto U.S. provisional application Ser. No. 60/818,502, filed on Jul. 6,2006, and to U.S. provisional application Ser. No. 60/818,500, filed onJul. 6, 2006 and to U.S. provisional application Ser. No. 60/818,499,filed on Jul. 6, 2006, and to U.S. provisional application Ser. No.60/760,363, filed on Jan. 20, 2006, and to U.S. provisional applicationSer. No. 60/751,323, filed on Dec. 19, 2005, and to U.S. provisionalapplication Ser. No. 60/751,322, filed on Dec. 19, 2005, and to U.S.provisional application Ser. No. 60/751,203, filed on Dec. 19, 2005, andto U.S. provisional application Ser. No. 60/751,202, filed on Dec. 19,2005, and to U.S. provisional application Ser. No. 60/735,857, filed onNov. 14, 2005, and to U.S. provisional application Ser. No. 60/734,260,filed on Nov. 8, 2005, and to U.S. provisional application Ser. No.60/734,259, filed on Nov. 8, 2005, and to U.S. provisional applicationSer. No. 60/734,258, filed on Nov. 8, 2005, and to U.S. provisionalapplication Ser. No. 60/733,168, filed on Nov. 4, 2005, and to U.S.provisional application Ser. No. 60/733,167, filed on Nov. 4, 2005, andto U.S. provisional application Ser. No. 60/730,006, filed on Oct. 26,2005, and to U.S. provisional application Ser. No. 60/730,005, filed onOct. 26, 2005, and to U.S. provisional application Ser. No. 60/730,004,filed on Oct. 26, 2005, and to U.S. provisional application Ser. No.60/730,003, filed on Oct. 26, 2005, and to U.S. provisional applicationSer. No. 60/726,662, filed on Oct. 17, 2005, and to U.S. provisionalapplication Ser. No. 60/726,658, filed on Oct. 17, 2005, the contents ofeach of which are hereby incorporated by reference in their entiretyinto this application.

FIELD OF THE INVENTION

This invention relates to the fields of molecular biology and oncology.Specifically, the invention provides molecular markers and therapeuticagents for use in the diagnosis and treatment of diseases, especiallycancer. In particular, the invention provides the following targets andmethods of using these targets: GFRa1, Claudin-4, ASCT2, CD166-ALCAM,CD55, TG2, CD49f, CD98, CD104, DPEP1, Tissue Factor (TF), Na—K ATPasebeta3, VIPR1, CD26, CXADR, PTK7, and MISTR (see Figures), which arecollectively referred to herein as “CAT” (cancer-associated targets).

BACKGROUND OF THE INVENTION

Cancer currently constitutes the second most common cause of death inthe United States, and cancer is difficult to diagnose and treateffectively. Accordingly, there is a need in the art for improvedmethods for detecting and treating various cancers. The presentinvention fulfills these needs and further provides other relatedadvantages, such as uses related to the treatment of other diseases.

Breast Cancer

Breast cancer is the primary killer of women. One in eight Americanwomen will develop breast cancer in her lifetime. An estimated 3 millionwomen in the U.S. today are living with breast cancer, which 2 millionhave been diagnosed with the disease and 1 million have the disease butdo not yet know it.

The incidence of breast cancer in the U.S. has more than doubled in thepast 30 years. In 1964, the lifetime risk was one in twenty. Today it'sone in eight. Breast cancer is the most commonly diagnosed cancer inwomen in both America and worldwide. One or more of a variety oftreatments such as surgery, radiotherapy, chemotherapy and hormonetherapy are used. The treatment course for a certain type of breastcancer is usually selected based on a various prognostic parameters, forexample, an analysis of specific tumor markers. (e.g. Porter-Jordan andLippman, Breast Cancer 8:73-100 (1994)). However, the use of establishedmarkers is insufficient to interpret the results and it still results inhigh mortality which is observed in breast cancer patients. Despiteconsiderable research into therapies for these and other cancers, breastcancer remains difficult to diagnose and treat effectively. Accordingly,there is a need in the art for improved methods for detecting andtreating such cancers.

Lung Cancer

Lung cancer is the second most prevalent type of cancer for both men andwomen in the United States and is the most common cause of cancer deathin both sexes. Lung cancer can result from a primary tumor originatingin the lung or a secondary tumor which has spread from another organsuch as the bowel or breast. The five-year survival rate for lung cancercontinues to be poor at 8-15% survival indicating a large unmet needwith regard to more effective treatments and better diagnosis. Theestimated total lung cancer deaths in the U.S. in 2003 are 157,200 andthe total estimated new cases in 2003 are 171,900. Primary lung canceris divided into three main types; small cell lung cancer; non-small celllung cancer; and mesothelioma. Small cell lung cancer is also called“Oat Cell” lung cancer because the cancer cells are a distinctive oatshape. There are three types of non-small cell lung cancer. These aregrouped together because they behave in a similar way and respond totreatment differently to small cell lung cancer. The three types aresquamous cell carcinoma, adenocarcinoma, and large cell carcinoma.Squamous cell cancer develops from the cells that line the airways.Adenocarcinoma also develops from the cells that line the airways.However, adenocarcinoma develops from a particular type of cell thatproduces mucus (phlegm). Large cell lung cancer has been thus namedbecause the cells look large and rounded when they are viewed under amicroscope. Mesothelioma is a rare type of cancer which affects thecovering of the lung called the pleura. Mesothelioma is often caused byexposure to asbestos.

Secondary lung cancer is cancer that has started somewhere else in thebody (for example, the breast or bowel) and spread to the lungs. Choiceof treatment for secondary lung cancer depends on where the cancerstarted. In other words, cancer that has spread from the breast shouldrespond to breast cancer treatments and cancer that has spread from thebowel should respond to bowel cancer treatments.

The stage of a cancer indicates how far a cancer has spread. Staging isimportant because treatment is often decided according to the stage of acancer. The staging is different for non-small cell and for small cellcancers of the lung.

Non-small cell cancer can be divided into four stages. Stage I is verylocalized cancer with no cancer in the lymph nodes. Stage II cancer hasspread to the lymph nodes at the top of the affected lung. Stage IIIcancer has spread near to where the cancer started. This can be to thechest wall, the covering of the lung (pleura), the middle of the chest(mediastinum) or other lymph nodes. Stage IV cancer has spread toanother part of the body.

Since small cell lung cancer can spread quite early in development ofthe disease, small cell lung cancers are divided into only two groups.These are: limited disease, that is cancer that can only be seen in onelung and in nearby lymph nodes; and extensive disease, that is cancerthat has spread outside the lung to the chest or to other parts of thebody. Further, even if spreading is not apparent on the scans, it islikely that some cancer cells will have broken away and traveled throughthe bloodstream or lymph system. To be safe, it is therefore preferredto treat small cell lung cancers as if they have spread, whether or notsecondary cancer is visible. Because surgery is not typically used totreat small cell cancer, except in very early cases, the staging is notas critical as it is with some other types of cancer. Chemotherapy withor without radiotherapy is often employed. The scans and tests done atfirst will be used later to see how well a patient is responding totreatment.

Procedures used for detecting, diagnosing, monitoring, staging, andprognosticating lung cancer are of critical importance to the outcome ofthe patient. For example, patients diagnosed with early lung cancergenerally have a much greater five-year survival rate as compared to thesurvival rate for patients diagnosed with distant metastasized lungcancer. New diagnostic methods which are more sensitive and specific fordetecting early lung cancer are clearly needed.

Lung cancer patients are closely monitored following initial therapy andduring adjuvant therapy to determine response to therapy and to detectpersistent or recurrent disease of metastasis. There is clearly a needfor a lung cancer marker which is more sensitive and specific indetecting lung cancer, its recurrence, and progression.

Another important step in managing lung cancer is to determine the stageof the patient's disease. Stage determination has potential prognosticvalue and provides criteria for designing optimal therapy. Generally,pathological staging of lung cancer is preferable over clinical stagingbecause the former gives a more accurate prognosis. However, clinicalstaging would be preferred were it at least as accurate as pathologicalstaging because it does not depend on an invasive procedure to obtaintissue for pathological evaluation. Staging of lung cancer would beimproved by detecting new markers in cells, tissues, or bodily fluidswhich could differentiate between different stages of invasion.

Pancreatic Cancer

The prognosis for pancreatic carcinoma is, at present, very poor.Pancreatic cancer displays the lowest five-year survival rate among allcancers. Such prognosis results primarily from delayed diagnosis, due inpart to the fact that the early symptoms are shared with other morecommon abdominal ailments. Despite the advances in diagnostic imagingmethods like ultrasonography (US), endoscopic ultrasonography (EUS),dualphase spiral computer tomography (CT), magnetic resonance imaging(MRT), endoscopic retrograde cholangiopancreatography (ERCP) andtranscutaneous or EUS-guided fine-needle aspiration (FNA),distinguishing pancreatic carcinoma from benign pancreatic diseases,especially chronic pancreatitis, is difficult because of thesimilarities in radiological and imaging features and the lack ofspecific clinical symptoms for pancreatic carcinoma.

Substantial efforts have been directed to developing tools useful forearly diagnosis of pancreatic carcinomas. Nonetheless, a definitivediagnosis is often dependent on exploratory surgery which is inevitablyperformed after the disease has advanced past the point when earlytreatment may be effected.

Colon Cancer

The prognosis of colon cancer is directly related to the degree ofpenetration of the tumor through the bowel wall and the presence orabsence of nodal involvement, consequently, early detection andtreatment are especially important. Currently, diagnosis is aided by theuse of screening assays for fecal occult blood, sigmoidoscopy,colonoscopy and double contrast barium enemas. Treatment regimens aredetermined by the type and stage of the cancer, and include surgery,radiation therapy and/or chemotherapy. Recurrence following surgery (themost common form of therapy) is a major problem and is often theultimate cause of death. In spite of considerable research intotherapies for the disease, colon cancer remains difficult to diagnoseand treat. In spite of considerable research into therapies for theseand other cancers, colon cancer remains difficult to diagnose and treateffectively. Accordingly, there is a need in the art for improvedmethods for detecting and treating such cancers.

Prostate Cancer

Prostate diseases include, for example, prostate cancer, as well asbenign prostatic hyperplasia (BPH) and prostatitis.

Prostate cancer is the most common non-skin cancer in the United States,where one in six American men develop prostate cancer during hislifetime. About 80% of prostate cancers are diagnosed in men over theage of 65. African-American men are 65% more likely to develop prostatecancer than Caucasian-American men and, furthermore, African-Americanmen tend to get more severe forms of prostate cancer and are more thantwice as likely to die from prostate cancer as are Caucasian-Americanmen. Approximately 25% of men with prostate cancer have a family historyof prostate cancer. The risk of prostate cancer doubles among men havinga first-degree relative with the disease; with two close relatives, aman's risk increases fivefold; and with three or more close relatives,the risk for developing prostate cancer is almost 100%.

Screening for prostate cancer is typically carried out using theprostate specific antigen (PSA) blood test and the digital rectal exam(DRE). The DRE and PSA test cannot confirm whether or not prostatecancer is present, but can indicate whether further testing is needed.If either the DRE or the PSA test indicates the presence of prostatecancer, a transrectal ultrasound (TRUS)-guided biopsy is typicallycarried out. A biopsy is the only way to confirm or diagnose thepresence of prostate cancer. During a biopsy, a TRUS is used to view andguide one or more needles into the prostate to take multiple smallsamples of tissue from different parts of the prostate. These tissuesamples are then examined for the presence of cancer in order togenerate a value known as a Gleason Grade, which characterizes theaggressiveness of a particular prostate tumor based on the microscopicappearance of the tissue. Prostate cancer is also staged, which is anassessment of the size and degree of metastases of prostate cancer,using either of two different staging systems (a traditional systemclassifies the disease into four clinical categories rated A through D;another system exists which is called TNM staging forTumor-Nodes-Metastases staging). The major treatment options forprostate cancer include hormonal therapy, surgery, radiation therapy,and chemotherapy. Early detection of prostate cancer increases thesuccess rate of these treatment options.

Stomach (Gastric) Cancer

Stomach diseases (also known as gastric diseases) include, for example,stomach cancer and ulcers (ulcers typically involve a break in thetissue lining the stomach).

Stomach cancer is the second most common cancer in the world, behindonly skin cancer. Stomach cancer occurs twice as often in men as womenand is the most prevalent carcinoma in East Asia, with the rate in Japanbeing more than seven times that in the United States and accounting forone-third of all cancer deaths in Japan. The average age of individualsafflicted by stomach cancer is 55 years of age.

Several different types of stomach cancer exist. Adenocarcinomas are themost common type of stomach cancer, accounting for 90-95% of malignanttumors of the stomach. Adenocarcinomas typically develop from theepithelial cells that form the innermost lining of the stomach's mucosa.Soft tissue sarcomas are another type of stomach cancer, and soft tissuesarcomas typically develop from the cells of the muscle layer of thestomach. Leiomyosarcoma is the most common type of soft tissue sarcomathat affects the stomach. Another type of sarcoma that can affect thestomach is a gastrointestinal stromal tumor (GIST). Lymphomas can alsoaffect the stomach, of which MALT (mucosa-associated lymphoid tissue)lymphoma is the most common type of lymphoma that affects the stomach.The stomach can also be affected by carcinoid tumors.

Stomach cancer can be diagnosed by an upper gastrointestinal (GI)series, which are x-rays of the esophagus and stomach taken after thepatient has drinken a barium solution. Alternatively, an endoscopy canbe carried out in which a tube is passed through the esophagus into thestomach and, if desired, a biopsy can be done to obtain a tissue samplefor laboratory analysis. Blood tests, chest x-rays, a CT scan of theabdomen, and a check for blood in the patient's stools may also becarried out. Treatment for stomach cancer can include a combination ofsurgery (termed “gastrectomy”), chemotherapy, and radiation therapy. Ifthe tumor is located close to the small intestine, a partial gastrectomymay be carried out in which a portion of the stomach is removed. If thetumor is located closer to the esophagus, a near-total gastrectomy maybe carried out.

Stomach cancer is staged based on how deep the tumor has penetrated thestomach lining, whether it has invaded surrounding lymph nodes, andwhether it has metastasized. The system most often used to stage stomachcancer in the United States is the American Joint Commission on Cancer(AJCC) TNM system. T indicates how far the tumor has grown within thestomach and into nearby organs, N indicates the degree to which thetumor has spread to lymph nodes, and M indicates the degree to which thetumor has metastasized to distant organs. In TNM staging, informationabout the tumor, lymph nodes, and metastasis is combined in a processcalled stage grouping in order to indicate a stage (represented bystages 0, I, IIA, IIB, III, IVA, and IVB). As the stage increases from 0to IV, the 5-year relative survival rates for patient's diagnosed withstomach cancer at each stage decreases from about 89% (for stage 0) toabout 7-8% (for stages IVA and IVB).

Kidney Cancer

The American Cancer Society estimates that there will be about 36,160new cases of kidney cancer (22,490 in men and 13,670 in women) in theUnited States in the year 2005, and about 12,660 people (8,020 men and4,640 women) will die from this disease. Kidney cancer (also referred toas renal cancer or renal cell carcinoma) mostly affects adults between50 and 70 years of age. If detected early, kidney cancer is curable.However, symptoms may not appear until the tumor has grown to a largesize or metastasized to other organs, at which point treatment isdifficult.

The 5-year survival rate for individuals diagnosed with kidney cancer isabout 90% for those individuals whose tumor is confined to the kidney,about 60% if it has only spread to nearby tissues, and about 9% if ithas spread to distant sites (American Cancer Society, Detailed Guide:Kidney Cancer. “What Are the Key Statistics for Kidney Cancer (RenalCell Carcinoma)?”).

The majority of kidney cancers are renal cell carcinomas (which accountsfor over 90% of malignant kidney tumors), also known as renaladenocarcinomas or clear cell carcinomas. There are five main types ofrenal cell carcinoma that are identified based on microscopicexamination of cell type: clear cell, papillary, chromophobe, collectingduct, and “unclassified.” Kidney cancers are also usually graded on ascale of 1 through 4 to indicate how similar the nuclei of the cancercells are to the nuclei of normal kidney cells (grade 1 renal cellcancers have cell nuclei that differ very little from normal kidney cellnuclei and generally have a good prognosis, whereas grade 4 renal cellcancer nuclei look considerably different from normal kidney cell nucleiand have a worse prognosis). In addition to grade, kidney cancers arealso characterized by stage, which describes the size of the cancer anddegree of metastasis. The most commonly used staging system is that ofthe American Joint Committee on Cancer (AJCC) (also referred to as theTNM system), although the Robson classification is an older system thatmay be occasionally used.

In additional to renal cell carcinomas, other types of kidney cancersinclude transitional cell carcinomas, Wilms tumors, and renal sarcomas.Wilms tumors are the most common type of kidney cancer in children andare extremely rare in adults. Benign (non-metastasizing) kidney tumorsinclude renal cell adenomas, renal oncocytomas, and angiomyolipomas(American Cancer Society, Detailed Guide: Kidney Cancer. “What Is KidneyCancer (Renal Cell Carcinoma)?”).

Risk factors for kidney cancer include the following: age older than 50years; male (men are twice as likely to get kidney cancer compared towomen); cigarette smoking; exposure to asbestos, cadmium, or organicsolvents; obesity; a high-fat diet; and von Hippel-Lindau disease (agenetic condition that has a high incidence of kidney cancer).

Symptoms of kidney cancer include hematuria (blood in the urine),abdominal or low back pain, weight loss, fatigue, anemia, fever, highblood pressure, and leg or ankle swelling.

In addition to a detailed medical history, physical examination, andlaboratory blood testing, diagnosis of kidney cancer may typicallyinclude a computed tomography (CT) scan, ultrasound, magnetic resonanceimaging (MRI), intravenous pyelography (a kidney test that utilizes dyeand x-rays), or arteriography (a test in which dye is applied to theblood vessels feeding the kidney). To detect metastatic disease, chestX-ray and bone scan may be implemented.

Treatment of kidney cancer in individuals whose tumor is confined to thekidney may involve surgical removal of the kidney (nephrectomy) andsurrounding tissue. Radiation therapy may be applied to treat pain andadvanced or metastatic kidney cancers or to help shrink a tumor that iscausing obstruction. Immunotherapy, such as interferon andinterleukin-2, may be used to boost the immune system in patients withadvanced kidney cancer (Journal of the American Medical Association,JAMA Patient Page: Kidney Cancer).

Liver Cancer

Liver diseases include, for example, liver cancer and liver cirrhosis.Liver cancers include malignant liver tumors such as hepatocellularcarcinoma (which is the most common type of liver cancer, accounting forabout 75% of primary liver cancers) and cholangiocarcinomas, as well asbenign liver tumors such as hemangioma, hepatic adenomas, and focalnodular hyperplasia. Among other risk factors (e.g., cirrhosis, such asfrom alcohol abuse), chronic infection with hepatitis B or hepatitis Cvirus is a significant liver cancer risk factor.

Furthermore, when cancer is found in the liver, it is often the casethat the cancer did not originate in the liver but rather spread to theliver from another cancer that began in a different part of the body.The liver is a common site of metastases for cancers in other organs(such as cancers of the lung, breast, colon, and rectum), particularlysince the liver receives blood from the abdominal organs via the portalvein. Tumor cells may detach from the primary cancer, enter thebloodstream or lymphatic channels, and travel to the liver where thetumor cells begin to grow independently.

Liver cancer is rarely diagnosed at an early stage because it usuallydoes not cause symptoms until the cancer is in its later stages and,because no screening tests exist, small tumors are difficult to detectby physical exams. Liver cancers can sometimes be detected using a bloodtest for alpha-fetoprotein (AFP). However, some tumors do not produceAFP in quantities significant enough to be detected until the tumor istoo large to be removed or has metastasized outside the liver. Inaddition to blood tests for AFP, other diagnostic techniques that may beused to detect liver cancer include ultrasound, CT scans, MRI,angiography, laparoscopy, and biopsy.

Once diagnosed, liver cancer is typically characterized by a stage usingRoman numerals I through IV, with a higher numeral indicating a moreserious cancer. Stage III is further sub-divided into A, B, and C.

The three main types of treatment for liver cancer are surgery,radiation therapy, and chemotherapy. Currently, surgery offers the onlychance of completely curing liver cancer. However, surgery can onlycompletely cure liver cancer if the cancer is small and can be entirelyremoved. Unfortunately, complete removal of most liver cancers is notpossible. Often the cancer is too large by the time it is detected, ispresent in many different parts of the liver, or has metastasized beyondthe liver. Also, many patients who have cirrhosis do not have enoughhealthy liver remaining for surgery to even be an option. Radiationtherapy may be used to shrink a liver tumor or to provide relief fromsymptoms such as pain, but it can not cure liver cancer and may notprolong survival for liver patients. With regards to chemotherapy, livercancer does not respond to most drugs. The most successful single drughas been doxorubicin (Adriamycin), however studies generally have notshown that chemotherapy prolongs survival for liver cancer patients.

Only a small fraction of liver cancers are detected at an early stageand can be successfully removed by surgery. Less than 30% of patientswho undergo surgery have their cancer completely removed. The overall5-year relative survival rate from liver cancer is approximately 7%.

Melanoma (Skin Cancer)

Skin cancer includes, for example, melanoma. Melanoma is a type ofcancer in which melanocytes (pigment cells) become cancerous. Melanomagenerally originates in the skin (cutaneous melanoma), however melanomacan sometimes originate in other areas of the body where melanocytes arepresent, such as the eyes, meninges, digestive tract, and lymph nodes.Other types of skin cancer include basal cell and squamous cell cancers.Melanoma is much more likely to metastasize and to be fatal than othertypes of skin cancer.

Melanoma is increasing in occurrence in the United States and worldwidefaster than any other cancer, with an approximately 3% annual increasein new cases. The risk for melanoma in the year 2000 was 1 in 74, andmelanoma is the most common cancer in individuals aged 20-30 and themost common cause of cancer death in women age 25-30 (and #2 cause ofdeath, after breast cancer, for women age 30-35). Melanoma accounts for5% of all skin cancers, but 71% of all skin cancer deaths. However, theearlier that melanoma is diagnosed, the better the prognosis forsurvival.

Thus, it is clear that early detection of cancer is desirable.Furthermore, it would also be desirable to identify individuals who havean increased risk of developing cancer in the future. Additionally,novel therapeutic agents are needed for treating cancer.

One promising method for early diagnosis of various forms of cancer isthe identification of specific biochemical moieties, termed targets,expressed differentially in cancerous cells. The targets may be eithercell surface proteins, cytosolic proteins, or secreted proteins.Antibodies or other biomolecules or small molecules that willspecifically recognize and bind to the targets in the cancerous cellspotentially provide powerful tools for the diagnosis and treatment ofthe particular malignancy.

GFRa1

GFRa1 is a cystein-rich glycosyl phosphatidylinositol (GPI)-linkedligand binding cell surface receptor. GFRa1 is a member of a family ofGFRa receptors that share a common signaling receptor tyrosine kinasesubunit c-RET. GFRa1 is the preferred binding partner ofglial-cell-line-derived neutrophic factor (GDNF). Following binding withGDNF, GDNF-GFRa1 forms a dimer that can interact with a kinase receptorcalled c-Ret. Activation of c-Ret triggers transphosphorylation ofspecific tyrosine residues and activation of intracellular signalingcascades that regulate cell survival, differentiation, proliferation,migration, chemotaxis, branching morphogenesis, neurite outgrowth, andsynaptic plasticity. Alternatively, GDNF-GFRa1 dimer can interact withNCAM to initiate signaling. Studies have shown that GDNF-activated N-CAMsignaling acts to promote CNS axon growth and Schwann cell migration.

Claudin-4

Claudin-4 is part of a superfamily (24 family members) of tight junction(TJ) related proteins. Claudin-4 is involved in cell-cell adhesion andhas an extracellular domain larger than 40 amino acids. Claudins are oneof three types of tight-junction cell adhesion proteins (occludin, JAM,claudins) and thought to be the most critical for constitutingtight-junction strands. Tight junctions form barriers at epithelial andendothelial cells. Tight junctions regulate cellular movement of waterand ions (intracellular sealing) and limit lateral diffusion of lipidsand proteins between the apical and basolateral membrane regions to formpolarized epithelia. Aggregated TJ proteins form networks of paired TJstrands between each plasma membrane at discrete sites of fusion ofplasma membranes of adjacent cells. Each TJ strand associates laterallywith another TJ strand in the plasma membrane of an adjacent cell toform a paired TJ strand.

Ion transport is charge- and size-selective with ion transport acrosstight-junctions. Cation selective transport is mainly by theparacellular pathway, and may be coupled with transcellular transport incertain situations. Activation of Na+-glucose transporters in theintestine is thought to alter structure and function of tight-junctions.With elevated glucose levels, absorption of glucose occurs by transportthrough tight-junctions when Na+-glucose transporters are saturated.

Macromolecular protein complexes form at tight-junctions. Proteins witha PDZ domain bind to the cytoplasmic surface of tight-junctions bydirect interaction with the carboxyl terminus of claudins. Thisfunctions to cross-link TJ strands to actin cytoskeleton, and plays arole in regulating paracellular transport across tight-junctions. Theseproteins also function as adaptor proteins to recruit signalingmolecules for activation of downstream signal transduction pathways, andplay a role in cell-matrix adhesion with formation of a complex atintegrin-based adhesion sites. For a further review, see Tsukita et al.,“Multifunctional strands in tight junctions”, Nat Rev Mol Cell Biol.2001 April; 2(4):285-93.

Altered permeability of tight junctions associated with multiplepathological states, including inflammation (e.g., inflammatory boweldisease) and tumorigenesis. Claudin-4 functions as a receptor forClostridium perfringens enterotoxin (CPE), which is known to injureintestinal epithelial cells and breast cancer cells by increasingmembrane permeability, thereby resulting in loss of osmotic equilibriumleading to cell death (Katahira et al. JCB 1999; Kominsky et al. Am JPathol 2004). Cytotoxic effects of CPE appears to be restricted toClaudin-4 expressing cells (demonstrated in pancreatic cancer), and hasbeen proposed as a novel treatment for Claudin-4 expressing solid tumors(Leder et al. Gastroenterology 2001).

Altered regulation of claudin-4 occurs in multiple cancers. For example,EGF signaling through the Ras signaling pathway increases proteinsynthesis of several claudins, including Claudin-4 (Singh et al. JBC2004). Claudins have been shown to activate pro-matrixmetalloproteinase-2 through direct interactions, and Claudin-4 recruitsmembrane-type matrix metalloproteinases on cell surfaces to high focalconcentrations of enzymes for activation of MMP-2 (Miyamori et al. JBC2001).

Claudin-4 is involved in cancer biology. For example, increasedclaudin-4 expression occurs in multiple tumor types [e.g., pancreaticcancer (Leder et al. Gastroenterology 2001), prostate cancer (Long etal. Cancer Res 2001), ovarian carcinoma (Santin et al. Int J Cancer2004; Hibbs et al. Am J Pathol 2004), and squamous cell carcinoma(keratinized tumors) (Morita et al. Br J Dermatol 2004)]. Overexpressionof Claudin-4 in pancreatic cancer correlates with decreased invasivenessin vitro (Boyden chamber) and in vivo (mouse lung colonization assay)(Michl et al. Cancer Res 2003). Reduced expression of claudin-4 andE-cadherin correlates with poor differentiation in gastric cancer (Leeet al. Oncol Rep 2005). Claudin-4 and claudin-3 are expressed in greaterthan 90% of breast carcinomas, but no correlation has been found withestrogen or progesterone receptor status or with tumor grade (Soini etal. Hum Pathol 2004).

Thus, Claudin-4 is involved in cell-cell adhesion, overexpression ofClaudin-4 is documented in the literature in several cancers, Claudin-4is overexpressed in greater than 90% of breast carcinomas, and Claudin-4is potentially involved in regulating metastasis and the invasivepotential of cancer cells.

ASCT2

ASCT2, also known as Neutral Amino Acid Transporter (NAAT), is a cellsurface transporter involved in cellular metabolism. ASCT2 has anextracellular domain that is larger than 100 amino acids in size.Antagonism of ASCT2 function stimulates apoptosis.

ASCT2, which is a member of the ASC amino acid transporter system, is aNa+ dependent transporter with high affinity for glutamine. ASCT2 alsotransports other zwitterionic amino acids such as serine, threonine,cysteine, alanine, and asparagine. Glutamine is an essential metabolicintermediate required for cellular growth. Glutamine requirements areincreased in rapidly dividing tumor cells (M A Medina. 2001. J Nutr131(9 Suppl):2539S-2542S).

Hepatoma-specific expression of ASCT2 is reported in the literature.ASCT2 is not expressed in normal liver tissue. In hepatocellularcarcinoma, glutamine uptake is 10-30 times faster than in normalhepatocytes. Antisense knockdown of ASCT2 expression resulted ininduction of apoptosis in hepatoma cells (B C Fuchs, et al. 2004. Am JPhysiol Gastrointest Liver Physiol 286:G467-G478).

Expression of ASCT2 in colorectal adenocarcinoma is reported in theliterature. ASCT2 protein was detected by western blot using MYZpolyclonal antibodies in colon tumor lysates. Immunohistochemistry (IHC)was performed on 63 colon tumor samples, with the following results:negative 41%, 1-25% of cell positive 24%, 26-50% of cells positive 13%,and greater than 50% of cells positive 22%. ASCT2 expression wasassociated with decreased patient survival (p=0.0002) (D Witte, et al.2002. Anticancer Res 22:2555-2558).

Expression of ASCT2 in prostate is reported in the literature. IHC wasperformed using MYZ polyclonal antibodies on 640 prostate samples[normal, benign prostatic hyperplasia (BPH), and adenocarcinoma]. Highlevel of ASCT2 expression was found in 49% normal, 25.8% BPH, and 25.3%adenocarcinoma. Significant decrease in ASCT2 expression in BPH andadenocarcinoma compared to normal prostate was observed. Higher ASCT2expression was associated with poor prognostic factors, aggressivebehavior, and poor survival (R Li, et al. 2003. Anticancer Res23:3413-3418).

CD166 (ALCAM)

ALCAM (interchangeably referred to as CD166) is a member of theimmunoglobulin superfamily IgSF. ALCAM is expressed in a subset ofactivated leukocytes, monocytes, fibroblasts, epithelial, and neuralcells. The extracellular region of ICAM (typically about 527 amino acidsin size) consists of five Ig-like domains. The two N-terminal domainsclosely resemble variable-type (V−) domains while the three followingdomains are more similar to the constant (C−) domains. ICAM has a shortintracellular domain that is typically about 32 amino acids in size.

ALCAM is involved in both homophilic adhesion and heterophilic adhesionto CD6, which is a member of the scavenger receptor cysteine-richsuperfamily. ALCAM may also bind NgCAM and HDL. CD6 is a member of thescavenger receptor cysteine-rich superfamily. CD6 is expressed onthymocytes, mature T-cells, and some B cells. Adherence of CD6+thymocytes to ALCAM-expressing thymic epithelial cells is important forthe differentiation and development of mature T cells. Interaction ofCD6+ mature T cells and CD166-expressing monocytic antigen presentingcells is important for T cell activation. T cell activation can beinhibited by addition of monomeric, soluble forms of CD6 or CD166.

ALCAM homophilic interactions are important in the homing ofhematopotietic stem cells on to stromal cells, thereby playing animportant role in the regulation of hematopoietic development.Clustering of ALCAM at the cell surface is regulated by the actincytoskeleton and the stabilization of this clustering may involve PKCa.

ALCAM contains a diSia epitope. diSia epitopes play an important role inneurite extension. Inhibition of this epitope using mAbs canspecifically inhibit neurite formation.

CD166−/− mice are viable, fertile, and display no gross externalmorphological defects, however axon fasciculation defects and retinaldysplasias are observed. No obvious defects in circulating lympohocytesare observed, but preliminary analysis reveals histologicalabnormalities of the spleen (Mol Cell Neurosci (2004) 59-69).

It has been demonstrated that ALCAM derived from an A375 melanoma linecontains GlcNAc beta 1-6 branched oligosaccharides, which is a sugarmoiety associated with metastatic potential (Melanoma Res (2004) 14479-485).

scFV, mAb, and CD6-Fc all induce ALCAM internalization into an ovariancarcimoma line. scFv-saporin immunotoxin selectively kills cell linesexpressing ALCAM (J Cell Sci (2005) Mar. 15).

Regarding the role of CD 166 (ALCAM) in tumor biology, CD 166 is amarker of tumor progression in primary malignant melanoma (MM). 4/38+ve(benign)—17/23+ve late stage (MM)—13/28 metastasis (Am J Path (2000) 15769-774). CD166 is overexpressed in colorectal carcinoma and correlateswith shortened patient survival (J Clin Path (2004) 57: 1160-1164).Expression of CD166 is associated with poor prognosis in bladder cancer(UroOncology (2003) 3 121-129). CD166 is upregulated in low-gradeprostate cancer and progressively lost in high-grade lesions (TheProstate (2003) 54 34-43). In two breast cancer studies, CD166expression appeared elevated in early stage tumors (PR+/ER+) (BreastCancer Res (2004) δ 478-487). Anti-ALCAM antibody decreasedproliferation in breast cancer cells and reduced adhesion (FASEB J(2004) 18 A330).

For a further review of ALCAM, see also J. Biol. Chem. (2004) 27955315-55323.

CD55

CD55 is a membrane-associated complement regulatory protein and has anextracellular domain that is typically larger than 300-400 contiguousamino acids. CD55 is involved in immune modulation and functions toprotect cells from bystander attack by blocking the complement cascade.

CD55 recognizes C4b and C3b fragments which are locally generated duringC4 and C3 activation. Interaction of DAF with cell-associated C4b andC3b polypeptides interferes with their ability to catalyze theconversion of C2 and factor B to enzymatically-active C2a and Bb andthereby prevents the formation of C4b2a and C3bBb, which are theamplification convertases of the complement cascade.

CD55 is expressed on all serum-exposed cells (red blood cells,leukocytes, endothelial cells and epithelial cells), and the solubleform is present in body fluids and extracellular matrix (serum level ˜30ng/ml) (C. Makidono et al., 2004. J Lab Clin Med 143:152-158).

CD55 is a possible ligand for CD97 (upregulated on most leukocytesduring activation), which may serve as an adhesion mechanism (J. Hamannet al., 1996. J Exp Med 184:1185-1189).

Complement regulatory proteins are commonly deregulated in tumor cells.Through over-expression of CD55, CD46, CD35 and/or CD59, tumor cells areable to protect themselves for complement-mediated lysis, which is amajor limitation to immunotherapeutic treatments.

CD55 is associated with poor prognostic indicator in colorectalcarcinoma. High CD55 levels are associated with a 24% survival rate, andlow CD55 levels are associated with a 50% survival rate (L. G. Durrant,et al., 2003. Cancer Immunol Immunother 52(10):638-42).

CD55 is upregulated in gastric carcinomas (T. Kiso et al., 2002.Histopathology 40:339-347). Also, loss of CD55 is associated withaggressive breast tumors (Z. Madjd, et al. 2004. Clin Cancer Res10(8):2797-803).

CD55 is cleaved from a GPI anchor in colorectal carcinomas and can bedetected in stool samples, and is therefore possibly useful as adiagnostic (M. Kawada et al. 2003. J Lab Clin Med 142:306-312).

Human IgM antibody (SC-1) against a tumor-specific form of CD55 has beenisolated from a gastric carcinoma patient. The antigenic site of SC-1 isan N-linked carbohydrate residue. SC-1 induces specific apoptosis ofgastric carcinoma cells both in vitro and in vivo (F. Hensel et al.,1999. Cancer Res 59:5299-5306). SC-1 was successfully used in a phaseI/II clinical study, showing induction of regression and apoptosis inprimary gastric carcinomas with minimal toxicity (H. P. Vollmers et al.,2004. J Clin Oncol 22:4070 (Abstract)).

Antibody that was raised against an osteosarcoma cell line was used forclinical imaging of over 300 patients with colorectal, gynecologic, andgastrointestinal lesions in the 1980s. The antibody detected lesions assmall as 1 cm³ in 70% of patients. The antigen for this antibody isCD55.

Anti-CD55 mAb significantly enhanced activity of Rituxan (Biogen IDEC,anti-CD20 or NHL) when used together in a cell-based study (Viragen).

Human anti-idiotypic antibody that mimics CD55 has been usedsuccessfully in over 200 colorectal and osteosarcoma patients (D. T. J.Buckley et al., 1995. Hum. Antibody Hybridoma 6:68-72).

Transglutaminase 2 (TG2)

Transglutaminase 2 (TG2) is a member of the transglutaminase family ofenzymes that catalyses Ca²⁺ dependent reactions, resulting in themodification of glutamine and lysine residues. TG2 contains a largeextracellular domain that is typically greater than 200 amino acids insize.

At membrane locations, TG2 can act as a G-protein to mediatetransmembrane signaling. Gh/TG2 couples a_(1b) and a_(1d)adrenoreceptors, thromboxane and oxytocin receptors to phospholipase C,mediating inositol phosphate production in response to agonistactivation. TG2 can also act as an isopeptidase in a Ca²⁺-dependentmanner. Thus, TG2 is able to modify major components of thecytoskeleton. TG2 is externalized from cells where it mediates theinteraction of integrins with fibronectin and cross-links proteins ofECM. This function has implications in adhesion and spreading. Undercertain conditions, TG2 translocates to the nucleus and functions as aG-protein or as a transamidase that cross-links histones. Thus, TG2 mayhave a role in chromatin modification or gene expression regulation.

CD49f

CD49f (integrin α6) is a cell surface receptor with an extracellulardomain that is typically larger than 1000 amino acids in size. Thisprotein functions as a receptor for laminin.

The α6 chain is found in only two heterodimeric combinations, α6β1 andα6β4. The α6β1 integrin (VLA-6) binds laminins-1, -2 and -4, while α6β4binds laminin-1 and, with higher affinity, laminin-5 (Eur. J. Biochem,1991; 199:425). The α6β4 integrin is found mainly in hemidesmosomes, andthe large cytoplasmic domain of the 134 integrin is important in theintegrity of these structures (EMBO J, 1990; 9:765). Mice lacking eitherα6 or β4 genes display perinatal lethality (Genes Dev, 1995; 9:1883).

CD49f contributes to breast carcinoma survival and progression (MolCells, 2004; 17:203) and is over-expressed in human esophagealcarcinomas (Int J. Oncol, 2000; 16:725) and human pancreatic carcinoma(Cancer Lett, 1997; 118:7). CD49f is also expressed in human pulmonarysquamous cell and adenocarcinomas (Hum Pathol, 1998; 29: 1208). Inhepatocellular carcinoma, CD49f exhibits differential display andmessenger RNA overexpression (Hepatology, 1995; 22: 1447). CD49f isassociated with a migratory and invasive phenotype in human prostatecarcinoma cells (Clin Exp Metastasis, 1995; 13: 481).

CD98

CD98 (4F2hc) (SLC3A2; solute carrier family 3 member 2 isoform A)belongs to a family of glycoprotein-associated amino acid transporters.CD98 has an extracellular domain that is typically greater than 400amino acids in size and functions as a sodium-independent transporterfor cellular uptake of large neutral amino acids. CD98 has beenimplicated in hematopoietic and osteoblast cell differentiation.

CD98 has been shown to be associated with CD147, ASCT2, and 131 integrin(CD29), which is involved in CD98-induced cell aggregation. Throughassociation with integrin, CD98 plays a role in cell adhesion,modulating the signaling for tumor cell proliferation and anchorageindependent growth. The association of CD98 with integrin α4β1 isinvolved in T-cell activation.

In its interaction with CD98, CD147 may act as an ancillary adhesionmolecule mediating cell-cell binding. CD147 may inhibit CD98 signalingfor homotypic aggregation by blocking CD98-induced tyrosinephosphorylation. Additionally, a CD98-CD147 complex may mediate cellproliferation as indicated by RNA interference (RNAi) knockdown

In several cancers such as salivary adenoid cystic carcinoma, oralsquamous cell carcinoma, squamous cell carcinoma of the larynx andgliomas, CD98 is overexpressed. Sequiterpene lactone cynaropicrininhibits activation of β1 integrin and CD98 aggregation bydownregulating expression of β1 integrin and CD147 and blockingdownstream the ERK signaling cascade and rearrangements of thecytoskeleton. The compound has anti-inflammatory and immunomodulatoryeffects and is cytotoxic and pro-apoptotic in cancer cells.

CD104 (β4 Integrin)

β4 integrin (CD104) is a Type I membrane protein comprising anextracellular domain typically greater than 500 amino acids and a largecytoplasmic domain typically greater than 1000 amino acids that contains4 fibronectin type III domains. Five alternate splice forms of β4integrin (β4A-β4E) have been identified. β4 integrin associates withintegrin α6 to form the heterodimer integrin α6/β4, which is a receptorfor laminin-5. α6/β4 uses laminin 5 anchoring filaments to attach anepithelium to the basal lamina to form hemidesmosomes. [J Cell Bio(1991) 113: 907-917]

Ligation of α6/β4 causes phosphorylation of the cytoplasmic tail of β4through activation of an integrin-associated Src family kinase, causingrecruitment of Shc and activation of Ras and PI-3K. The phosphorylationof β4 causes disruption of hemidesmosomes [J Biol Chem (2001) 276:1494-1502], [Cancer Cell (2004) δ: 471-483]. The N-terminus of the β4cytoplasmic domain (up to amino acid residue 1355) is involved inadhesion. The C-terminal portion of the β4 tail contains 5 tyrosinephosphorylation sites, including those needed for recruitment of Shc andPI-3K. Targeted deletion of the C-terminal portion of the β4 tailinhibited signaling through ERK and AKT, but not adhesion to laminin 5and assembly of hemidesmosomes.

β4 integrin promotes endothelial cell migration and invasion, with theβ4 substrate domain inducing the nuclear accumulation of ERK and NF-κBduring endothelial cell migration (in vitro) and angiogenesis (in vivo).The β4 substrate domain has been identified as promoting tumor invasionand angiogenesis [Cancer Cell (2004) δ: 471-483].

Signaling by α6/β4 has been shown to promote bFGF and VEGF inducedangiogenesis [Cancer Cell (2004) δ: 471-483]. Signaling by α6/β4 alsopromotes adhesion of keratinocytes through phosphorylation of PKB/Akt [JInvest Dermatol 123:444-451].

β4 integrin knockout mice exhibit a lack of hemidesmosomes and havesevere junctional epidermolysis bullosa (epidermal blistering). β4 nullmice were unable to survive more than a few hours after birth [J CellBio (1996) 134: 559-572]. In autosomal dominant polycystic kidneydisease, kidney cysts demonstrate an overexpression of β4 integrin [Am JPathol (2003) 163: 1791-1800].

By mediating tumor cell adhesion to endothelial CLCA2 (associated withcolonization of the lung by breast cancer cells), β4 integrin isinvolved in the metastasis of breast cancer to the lung [J Biol Chem(2001) 276: 25438-35446]; [J Biol Chem (2003) 278: 49406-49416]. β4integrin ligation to mCLCA1 (homolog to hCLCA2) activates focal adhesionkinase and mediates early metastatic growth of B16-F10 melanoma cells inthe lung [J Biol Chem (2002) 277: 34391-34400]. β4 integrin also causesselective apoptosis in endothelial cells bound to tumor cells throughactivation of chloride channels. Endothelial cells incubated with β4integrin undergo apoptosis [J Biol Chem (2001) 276: 25438-35446].

DPEP1

Dipeptidase 1 (DPEP1) (Swiss Prot Accession Number: P16444) is aGPI-anchored cell surface glycoprotein (homodimer) (J. Mol. Bio. (2002)321: 177-184). DPEP1 has an extracellular domain that is typically about369 amino acids in size.

DPEP1 is a zinc-containing enzyme that hydrolyzes various dipeptides,antibiotics (b-lactams), and leukotrienes. DPEP1 is implicated in renalmetabolism of glutathione and it conjugates. DPEP1 is expressed in thebrush-border region of the proximal tubules of the renal cortex(Biochem. J. (1989) 257: 361-367). Cilastatin, an inhibitor of DPEP1, iscommonly delivered with Imipenem, a b-lactam antibiotic, to inhibitbreakdown in the kidney (J. Antimicrob. Chemother. (1983) 12: 1-35).

DPEP1 was determined to be >20-fold over-expressed in colon adenomas andcarcinomas by SAGE analysis and localized to the epithelium ofcolorectal tumors by ISH (Cancer Research (2001) 61: 6996-7001). DPEP1showed 2-fold or greater over-expression by relative RT-PCR in colontumors compared to normal colon in 82% of patients tested, andover-expression observed in all stages of disease. DPEP1 was detected byRT-PCR in disseminated tumor cells purified from the blood andintra-operative lavage samples of colorectal cancer patients (CancerLetters (2004) 209: 67-74).

Tissue Factor (TF)

Tissue factor (TF) is a cell surface glycoprotein with a largeextracellular domain (typically greater than 200 amino acids in size).TF is the primary cellular initiator of blood coagulation where itserves as the cellular receptor for Factor VII. Signaling events linkedto TF-Factor VIIa interaction can lead to tumor cell proliferation,transcriptional changes, altered cell-shape, and migration/adhesion.

TF is typically not expressed in cells that are in direct contact withthe blood. TF is expressed in extravascular tissue, in fibroblasts andsmooth muscle cells, where it serves as a haemostatic envelope outsidethe vasculature, poised to activate coagulation upon vascular injury. Invascular endothelial cells and monocytes, TF is typically absent, but TFis rapidly induced in response to inflammatory stimuli such as bacterialliposaccharide and inflammatory cytokines. Increased intravascularlevels of TF have been reported in diverse pro-thrombotic syndromes suchas myocardial infarction and sepsis.

Patients with malignant diseases are predisposed to hypercoagulation.Trousseau first reported the increased frequency of thrombosis inpatients with gastrointestinal cancers in 1865, and subsequently thishypercoagulable state has been associated with TF. Correlation betweenelevated expression of TF and both advanced stages of malignancy and/orpoorly differentiated tumors has been reported in several cancersincluding pancreatic, breast, colorectal, NSCLC, prostate, and glioma.Elevated expression of TF in tumors has also been correlated with othernon-favorable prognostic indicators such as increased angiogenesis andmulti-drug resistance. An alternatively spliced secreted form of TF hasbeen detected in plasma that has elevated expression levels in normalsamples (˜50 pg/mL) compared with tumors (50-350 pg/mL) in the plasma ofbreast cancer patients. For further information regarding TF, seeBogdanov et al., “Alternatively spliced human tissue factor: acirculating, soluble, thrombogenic protein”, Nat. Med. (2003) 9:458-462.

Transfection of TF promoted metastasis in a melanoma mouse model,indicating a role for the cytoplasmic domain (Proc Natl Acad Sci USA(1995) 92:8205-9). Introduction of TF into a pancreatic adenocarcinomacell line lead to both increased tumor cell invasion in vitro andprimary tumor growth in vivo (Br J. Surg. (1999) 86:890-4). TF knockdownby RNAi suppressed invasiveness of a pancreatic cell line (BxPC3) invitro (Clin Cancer Res (2005)11:2531-2539). Humanized mAb (CNTO 859,860) reduced metastasis (MDA-MB-231) to the lung from tail-injected1000×, and also reduced tumor growth in a SubQ model (Centocor) (Journalof Immunotherapy. (2004) 27(6):S10). Anti-TF mAb (H36) abolishedprostate (DU145) and reduced breast (MDA-MB-435) metastasis to the lung(Sunol/Dow) (Journal Thrombosis and Haemostasis (2003) 1 Supplement 1July: # OC308).

NA/K ATPase Beta3

NA/K ATPase beta3 is a transporter involved in cellular metabolism andregulates a variety of transport functions in epithelial cells. NA/KATPase beta3 has an extracellular region that is typically larger than200 amino acids in size.

Blocking NA/K ATPase beta3 has been reported to sensitize tumor cells topro-apoptotic stimuli.

Vasoactive Intestinal Polypeptide Receptor 1 (VIPR1)

Vasoactive intestinal polypeptide receptor 1 (VIPR1) belongs to theG-protein coupled receptor 2 family and has an extracellular domain thatis typically greater than 100 amino acids in size. VIPR1 is involved inpulmonary and gastrointestinal vascular smooth-muscle relaxation, andVIPR1 ligand (VIP) analogs are in clinical development asbronchodilators for respiratory diseases such as COPD and asthma.

CD26

CD26 (dipeptidylpeptidase 4, DPP4, or adenosine deaminase complexingprotein 2) is a type II cell surface serine exopeptidase that has anextracellular domain that is typically greater than 700 amino acids insize and which also exists in soluble form. CD26 is implicated invarious biological processes including cell-matrix interactions, T-cellactivation, inflammation, and regulating insulin secretion. Inhibitorsof CD26 are in clinical development for type 2 diabetes.

CXADR

CXADR (coxsackie virus and adenovirus receptor; Swiss-Prot AccessionNumber: P78310) is a type I membrane receptor and a member of theimmunoglobulin superfamily (Science (1997) 275; 1320-1323). CXADR has anextracellular domain that is typically larger than 200 amino acids insize. CXADR is a component of the epithelial apical junction complexthat is essential for the tight junction integrity (J Biol Chem (1999)274; 10219-10226). CXADR recruits intracellular PDZ domain-containingprotein LNX (Ligand-of-Numb Protein-X) to intercellular contact sites (JBiological Sci (2003) 278; 7439-7444). CXADR may function as ahomophilic cell adhesion molecule (Molecular Brain Research (2000) 77;19-28). CXADR is involved in transepithelial migration of PMN throughadhesive interactions with JAML located in the plasma membrane of PMN(Mol Biol Cell (2005) 16; 2694-703). CXADR functions as a receptor forgroup B coxsackieviruses and subgroup C of adenoviruses (AD2 and AD5);susceptibility to infection has been correlated with membrane expressionlevel (Proc. Natl. Acad. Sci. (1997) 94; 3352-56). CXADR knockout miceexhibited embryonic lethal phenotype associated with cardiac defects(Genesis (2005) 42; 77-85).

Over expression of CXADR has been observed in osteosarcomas andmalignant thyroid tumors (Cancer Sci (2003) 94; 70-75; Thyroid (2005)15; 977-87). CXADR is involved in mediating tumor formation in lungcancer cells; a CXADR antisense plasmid vector abrogated xenograftsmediated by high expressing lung cancer cells and inhibited soft agarcolony formation (Cancer Res (2004) 64; 6377-80). CXADR expression isenhanced after transition from preneoplastic precursor lesions toneoplastic mammary cancer outgrowth in a syngenic mouse tumor model(Clin Cancer Res (2005) 11; 4316-20). In a 3D tissue culture model ofbreast cancer cells, disruption of polarity and integrity, as inmalignant transformation, can lead to up-regulation of CXADR (Proc.Natl. Acad. Sci. (2003) 100, 1943-1948). CXADR overexpression in ovarianand cervical cancer cell lines enhanced cell survival by protectingagainst apoptosis (Clin Cancer Res (2005) 11; 4316-20). Expression ofCXADR in gastrointestinal cancers correlated with tumor differentiation(Cancer Gene Ther (2006) Epub). Loss of CXADR expression associated withadvanced bladder cancer (Urology (2005) 66; 441-6). Over-expression ofCXADR in an ovarian cancer cell line inhibited cell migration (Exp CellRes (2004) 298; 624-31). Expression of CXADR decreased in primaryprostate cancer but is highly expressed upon metastasis (Cancer Res(2002) 62; 3812-8).

PTK7

Protein tyrosine kinase 7 (PTK7) is a transmembrane glycoproteincontaining RTK consensus sequences that may function in kinasesignaling, cell adhesion and signal transduction, and planar cellpolarity (PCP) pathways. PTK7 has an extracelluar domain that is largerthan 600 amino acids in size.

Macrophage-Stimulating Protein Receptor Precursor (MISTR)

Macrophage-stimulating protein receptor precursor (MISTR), also known asMST1R, MSP receptor, p185-Ron, CDW136, and CD136 antigen, is a receptortyrosine kinase that has an extracellular domain that is typicallylarger than 800 amino acids in size.

SUMMARY OF THE INVENTION

A diseased, e.g. malignant, cell often differs from a normal cell by adifferential expression of one or more proteins. These differentiallyexpressed proteins, and suitable fragments thereof, are useful asmarkers for the diagnosis and treatment of the disease. The presentinvention provides the following targets and methods of using thesetargets: GFRa1, Claudin-4, ASCT2, CD166-ALCAM, CD55, TG2, CD49f, CD98,CD104, DPEP1, Tissue Factor (TF), Na—K ATPase beta3, VIPR1, CD26, CXADR,PTK7, and MISTR (see Figures), which are collectively referred to hereinas “CAT” (cancer-associated targets). Each of these targets isassociated with specific types of cancers in particular, as shown in theFigures and described in section 14 of the Examples section (“Summary ofexperimental validation”).

Based on the finding that CAT are differentially expressed in diseasecells, particularly in cancer, in comparison to normal cells, thepresent invention provides methods and compositions for diagnosing andtreating diseases, particularly cancer, using CAT as a target.

In the context of the present invention, the differentially expressedCAT proteins (SEQ ID NOS:1-4, 9, 11-12, 16-19, 24-36, 51-59, 69-74,81-85, 91-102, 115-116, 119-121, 127-134, 144-145, 148-149, 152-159,168-174, and 182-183) and suitable fragments thereof, and nucleic acidsencoding said proteins (SEQ ID NOS:5-8, 10, 13-15, 20-23, 37-50, 60-68,75-80, 86-90, 103-114, 117-118, 122-126, 135-143, 146-147, 150-151,160-167, 175-181, 184-185) and suitable fragments thereof, are referredto herein as CAT proteins, CAT peptides, or CAT nucleic acids, andcollectively as CAT.

The CAT proteins of the present invention may serve as a target for oneor more classes of therapeutic agents, including antibody therapeutics.CAT proteins of the present invention are useful in providing a targetfor diagnosing a disease, or predisposition to a disease mediated by thepeptide, particularly cancer. Accordingly, the invention providesmethods for detecting the presence, or levels of, a CAT protein of thepresent invention in a biological sample such as tissues, cells andbiological fluids isolated from a subject.

The diagnosis method may detect CAT nucleic acids, proteins, peptides,and fragments thereof, that are differentially expressed in diseases ina test sample, preferably in a biological sample.

The further embodiment includes but is not limited to, monitoring thedisease prognosis (recurrence), diagnosing disease stage, preventing thedisease and treating the disease.

Accordingly, the present invention provides a method for diagnosing ordetecting a disease (particularly cancer) in a subject comprising:determining the level of CAT in a test sample from said subject, whereina differential level of said CAT in said sample relative to the level ina control sample from a healthy subject, or the level established for ahealthy subject, is indicative of the disease. The test sample includesbut is not limited to a biological sample such as tissue, blood, serumor biological fluid.

The diagnostic method of the present invention may be suitable formonitoring the disease progression or the treatment progress.

The diagnostic method of the present invention may be suitable for otherepithelial-cell related cancers, such as lung, colon, prostate, ovarian,breast, bladder renal, hepatocellular, pharyngeal, and gastric cancers.The present invention further provides antagonists to CAT proteins orpeptides and pharmaceutical compositions that comprise the antagonistand a suitable carrier. The antagonist may be used for treating thedisease. Preferably, the antagonist is an antibody that specificallybinds to a CAT protein or peptide. In another preferred embodiment, theantagonist may be a small molecule that inhibits the function or levelsof CAT, or an inhibitory nucleic acid molecule, such as an RNAi orantisense molecule against a CAT nucleic acid.

The present invention provides additionally a pharmaceutical compositioncomprising an antagonist to a CAT of the present invention, and apharmaceutically acceptable excipient, for treating a disease,particularly cancer.

The present invention further provides a method for treating a disease,particularly cancer, comprising administering to a patient in need ofsaid treatment a therapeutically effective amount of the pharmaceuticalcomposition.

The present invention further provides a method for screening for agentsthat modulate CAT protein activity, comprising the steps of (i)contacting a candidate agent with a CAT protein, and (ii) assaying forCAT protein activity, wherein a change in said activity in the presenceof said agent relative to CAT protein activity in the absence of saidagent indicates said agent modulates said CAT protein activity.Candidate agents include but are not limited to protein, peptide,antibody, nucleic acid such as antisense RNA, RNAi fragments, smallmolecules. RNAi is particularly effective at suppressing geneexpression, and is therefore useful for blocking or limiting productionof a CAT protein, such as for treating cancer or other diseases.

The screening method may also determine a candidate agent's ability tomodulate the expression level of a CAT protein or nucleic acid. Themethod comprises (i) contacting a candidate agent with a system that iscapable of expressing a CAT protein or CAT mRNA, (ii) assaying for thelevel of a CAT protein or CAT mRNA, wherein a specific change in saidlevel in the presence of said agent relative to a level in the absenceof said agent indicates said agent modulates said CAT level.

The present invention further provides a method to screen for agentsthat bind to a CAT protein, comprising the steps of (i) contacting atest agent with a CAT protein and (ii) measuring the level of binding ofagent to said CAT protein.

DESCRIPTION OF THE SEQUENCE LISTING

The Sequence Listing discloses exemplary protein and nucleic acidsequences for each CAT (Cancer-Associated Target). Specifically, theSequence Listing discloses amino acid sequences of CAT proteins andnucleic acid sequences of CAT transcripts that encode these CATproteins, as set forth in the following table:

Cancer-Associated Target Protein SEQ ID NO Transcript SEQ ID NO GFRa11-4 5-8 Claudin-4 9 10 ASCT2 11-12 13-15 CD166-ALCAM 16-19 20-23 CD5524-36 37-50 TG2 51-59 60-68 CD49f 69-74 75-80 CD98 81-85 86-90 CD104 91-102 103-114 DPEP1 115-116 117-118 Tissue Factor (TF) 119-121 122-126Na-K ATPase beta3 127-134 135-143 VIPR1 144-145 146-147 CD26 148-149150-151 CXADR 152-159 160-167 PTK7 168-174 175-181 MISTR 182-183 184-185

The Sequence Listing is hereby incorporated by reference pursuant to 37CFR 1.77(b)(11).

DESCRIPTION OF THE FIGURES

GFRa1

FIG. 1. GFRa1 is Over-Expressed in Breast and Renal Cancer.

FIG. 2. Prevalence of GFRa1.

FIG. 3. IHC Analysis Reveals GFRa1 staining on Breast Cancer samplesdoes not correlate with ER, PR or HER2Status.

FIG. 4. GFRa1 Expression in Breast and Kidney Cell Lines and Tumors.

FIG. 5. GFRa1 mRNA Expression Analysis in Multiple Tumor Tissues.

FIG. 6. Knockdown of GFRa1 mRNA Inhibits Proliferation in Lung andKidney Cancer Cells.

FIG. 7. Knockdown of GFRa1 mRNA Induces Apoptosis in Kidney CancerCells.

FIG. 8. Knockdown of GFRa1 mRNA Induces Apoptosis and InhibitsProliferation in Caki-1 Kidney Cancer Cells.

FIG. 9. Ligand of GFRa1 (GDNF) Increases Proliferation of GFRa1 PositiveMCF-7 Breast Cancer cells.

FIG. 10. GFRa1 Kinase Binding Partner (Ret) Kinase Inhibitors InhibitProliferation Induced by GFRa1 Ligand (GDNF) in MCF-7 Cells.

FIG. 11. GDNF Ligand IHC. Antibody to GFRa1 Ligand (GDNF) was evaluatedin immunohistochemistry at a concentration of 5 ug/ml on humanmulti-cancer slides. Sixty percent of pancreatic carcinomas, 40% ofbreast carcinomas, 30% of ovarian carcinomas, and 20% of lung non-smallcell carcinomas showed faint staining of the majority of neoplasticcells. Ten percent of colon and prostate carcinomas showed some faintstaining. Within the faint positive carcinomas, membrane staining wasfrequently observed. None of the tumors showed moderate or strongstaining with this antibody.

FIG. 12. Ret Kinase Binding Partner IHC. Antibody RDS-AF1485, a goatpolyclonal antibody to non-phosphorylated GFRa1 Kinase Binding Partner(Ret) was evaluated in immunohistochemistry on human multi-cancersections at 5 ug/ml. Fifty percent of prostate carcinomas and 40% ofbreast carcinomas showed faint to moderate staining in the majority oftumor cells. Forty percent of pancreatic carcinomas showed faintstaining, with adjacent islets of Langerhans showing moderate staining.Ten percent of colon carcinomas showed faint staining. Ten percent ofnon-small cell carcinomas of the lung showed faint staining, and ovariancarcinomas were negative. Staining within the carcinoma cells was mostlycytoplasmic, although occasional membrane-associated staining could beseen in subsets of moderately positive cells.

FIG. 13. Phosphorylated Ret Kinase Binding Partner IHC. One commercialantibody (a rabbit polyclonal to a peptide phosphorylated at residueTyr905) was evaluated in immunohistochemistry on human multi-cancersections at 1:25 and 1:50 dilution. At a dilution of 1:25, 90% of breastcarcinomas, 50% of pancreatic and 30% of ovarian carcinomas showedmostly faint to occasionally moderate staining. Faint staining was seenin 60% of colon carcinomas, 50% of prostate and 40% of lung non-smallcell carcinomas. At this dilution, artifactual nuclear staining was alsopresent. At a dilution of 1:50, 40% of breast, 30% of pancreatic, andprostate, and 10% of carcinomas of the colon, lung, and ovary showedfaint staining. Nuclear staining was less prevalent at this antibodydilution. Scores in the following sections reflect this antibodydilution. This antibody showed mostly cytoplasmic staining, orcytoplasmic and nuclear staining. At both dilutions, moderate to strongstaining was seen in residual pancreatic islets of Langerhans.

FIG. 14. Expression in Breast Cancer Specimens.

FIG. 15. Evaluation of Level and Homogeneity of GFRa1 Expression inTumor Tissues: 1st Ab.

FIG. 16. Evaluation of Level and Homogeneity of GFRa1 Expression inTumor Tissues: 2nd Ab.

FIG. 17. GFRa1 Ligand is Expressed in MCF-7 cells.

FIG. 18. Overexpression of mRNA for GFRa1 in Breast Tumors.

FIG. 19. Overexpression of mRNA for GFRa1 Kinase Binding Partner inBreast Tumors.

FIG. 20. Overexpression of mRNA for GFRa1 Ligand in Breast Tumors.

FIG. 21. Both GFRa1 and it's Kinase Binding Partner are Expressed inMCF7 and HCC1937 Breast Cancer Cells.

FIG. 22. GFRa1 Ligand Increases Proliferation of GFRa1 Positive MCF-7Breast Cancer cells.

FIG. 23. Ligand of GFRa1 Increases Proliferation of GFRa1 Positive MCF-7Breast Cancer cells.

FIG. 24. GFRa1 is Expressed in ACHN and Caki 1 Kidney Cell lines.

FIG. 25. GFRa1 Peptide Blocks 20 ng/ml GDNF (GFR Ligand) Mediated MCF-7cell Proliferation.

FIG. 26. GFRa1 Expression in Breast Cell Lines and Tumors.

FIG. 27. GFRa1 Kinase Binding Partner but not GFRa1 is Expressed inASPC-1 and BXPC-3 Pancreatic Cancer Cells.

FIG. 28. Heat Denatured GFRa1 Ligand Does not Induce MCF-7 cellProliferation.

FIG. 29. GFRa1 Ligand Binding on MCF-7 Breast Cancer Cells.

FIG. 30. GFRa1 Ligand mAb Blocks Binding of GFRa1 Ligand.

FIG. 31. GFRa1 Ligand-Mediated MCF-7 cell Proliferation Is Blocked byNeutralizing anti-GFRa1 Ligand Ab.

FIG. 32. Effect of GFRa1 Ligand/GFRa1 Antagonists on MCF-7 Proliferationin Complete Growth Medium (No Exogenous GFRa1 Ligand).

FIG. 33. GFRa1 Ligand (GDNF) Increases Proliferation of MCF-7 BreastCancer cells in a Statistically Significant Manner (n=3).

FIG. 34. mRNA sequence of GFRa1, indicating siRNA target regions.

Claudin-4

FIG. 35. Claudin-4 is Expressed at Cell Surface in Multiple Tumor Types,as indicated by IHC.

ASCT2

FIG. 36. ASCT2 is Over-expressed in Multiple Tumor Types.

FIG. 37. ASCT2 mRNA Expression Analysis in Pancreatic Tumors.

FIG. 38. Knockdown of ASCT2 mRNA Inhibits Proliferation in Pancreatic,Colon and Breast Cancer Cells.

FIG. 39. Knockdown of ASCT2 mRNA Inhibits Proliferation in ASPC-1Pancreatic Cancer Cells.

FIG. 40. Knockdown of ASCT2 mRNA Inhibits Proliferation in HT29 ColonCancer Cells.

FIG. 41. ASCT2 mRNA sequence, indicating siRNA target regions.

CD166 (ALCAM)

FIG. 42. CD166 is Overexpressed in Multiple Tumor Types.

FIG. 43. CD166 Overexpression in Tissues by Q-FACS.

FIG. 44. CD166 Overexpression in Cell Lines by Q-FACS.

FIG. 45. CD166 mRNA Expression—Breast Normal/Tumor Panel.

FIG. 46. CD166 mRNA Expression—Colon Normal/Tumor Panel.

FIG. 47. CD166 mRNA Expression Matched Colon Normal/Tumor Panel.

FIG. 48. RNAi Knockdown of CD166 mRNA Inhibits Proliferation in MultipleCancer Cells.

FIG. 49. RNAi Knockdown of CD166 mRNA Induces Apoptosis in Colon andGastric Cancer Cells.

FIG. 50. RNAi Knockdown of CD166 mRNA Inhibits Proliferation and InducesApoptosis in HT29 Colon Cancer Cells.

FIG. 51. RNAi Knockdown of CD 166 mRNA Inhibits Proliferation in ASPC-1Pancreatic Cancer Cells.

FIG. 52. RNAi Knockdown of CD166 mRNA Inhibits Proliferation and InducesApoptosis in Caki-1 Kidney Cancer Cells.

FIG. 53. RNAi Knockdown of CD166 mRNA Inhibits Proliferation in AGSGastric Cancer Cells.

FIG. 54. CD166 siRNA in Combination with Gemzar Increases Apoptosis ofBXPC-3 Pancreatic Cancer Cells.

FIG. 55. Saporin-Conjugated 2nd Ab+CD166 mAb Induces Cell Death in CD166Positive HCC1954 Breast Cells.

FIG. 56. Saporin-Conjugated 2nd Ab+CD166 mAb Does Not Induce Cell Deathin CD166 Negative HCC1937 Breast Cells.

FIG. 57. Saporin-Conjugated 2nd Ab+CD166 mAb Induces Cell Death in CD166Positive HCC1954 Breast Cells.

FIG. 58. Saporin-Conjugated 2nd Ab+CD166 mAb Does Not Induce Cell Deathin CD166 Negative HCC1937 Breast Cells.

FIG. 59. Saporin-Conjugated 2nd Ab+CD166 mAb Induces Cell Death in CD166Positive HCC1954 Breast Cells.

FIG. 60. Saporin-Conjugated 2nd Ab+CD166 mAb Does Not Induce Cell Deathin CD166 Negative HCC1937 Breast Cells.

FIG. 61. mRNA Sequence of CD166 (ALCAM), indicating siRNA targetregions.

CD55

FIG. 62. CD55 is Over-expressed in Multiple Tumor Types.

FIG. 63. QFACS Confirms Over-Expression of CD55 on the Surface of ColonTumors.

FIG. 64. QFACS Analysis Confirms that CD55 is Differentially Expressedin Colon Tumor Tissues.

FIG. 65. CD55 Expression Analysis by QFACS—Hematopoietic Cells vs. ColonTissue.

FIG. 66. CD55 mRNA Expression Analysis in Tumor Tissues.

FIG. 67. Knockdown of CD55 mRNA Inhibits Proliferation in Colon andProstate Cancer Cells.

FIG. 68. Knockdown of CD55 mRNA Inhibits Proliferation in HCT116 ColonCancer Cells.

FIG. 69. CD55 Expression in PBMC and Bone Marrow.

FIG. 70. CD55 Expression Analysis by QFACS—Hematopoietic Cells vs. ColonCell Lines.

FIG. 71. CD55 Expression Analysis by QFACS—Hematopoietic Cells vs.Pancreatic Cell Lines.

FIG. 72. CD55 Expression in Hematopoietic Cells.

FIG. 73. CD55 mRNA Overexpression—Pancreas Cell Line Panel.

FIG. 74. CD55 mRNA is Overexpressed in Colon Tumor Tissue.

FIG. 75. CD55 mRNA is Overexpressed in Colon Tumor Tissue.

FIG. 76. CD55 mRNA is Overexpressed in Colon Tumor Tissue.

FIG. 77. CD55 mRNA Expression in Pancreatic Tumor Tissues.

FIG. 78. CD55 mRNA Expression in Colon Tumor Tissues.

FIG. 79. CD55 qFACS Data Summary.

FIG. 80. CD55 qFACS Data Summary.

FIG. 81. CD55 is Over-expressed in Multiple Tumor Types.

FIG. 82. mRNA sequence of CD55, indicating siRNA target regions.

TG2

FIG. 83. TG2 is Over-Expressed in Multiple Tumor Types.

FIG. 84. TG2 mRNA Expression Analysis in Multiple Tumor Tissues.

FIG. 85. Knockdown of TG2 mRNA Inhibits Proliferation in Pancreas, Lungand Colon Cancer Cells.

FIG. 86. Knockdown of TG2 mRNA Inhibits Proliferation in HT29 ColonCells.

FIG. 87. Knockdown of TG2 mRNA Inhibits Proliferation in Calu1 LungCells.

FIG. 88. Knockdown of TG2 mRNA Inhibits Proliferation in BXPC-3 PancreasCells.

FIG. 89. mRNA sequence of TG2, indicating siRNA target regions.

CD49f

FIG. 90. CD49f is Over-Expressed in Multiple Tumor Types by IHC.

FIG. 91. CD49f is Over-expressed in Colon Tumors Relative to PBMC andBone Marrow.

FIG. 92. CD49f mRNA Expression Analysis in Tumor Tissues.

FIG. 93. Knockdown of CD49f mRNA Inhibits Proliferation in Lung, Colonand Gastric Cancer Cells.

FIG. 94. Knockdown of CD49f mRNA Inhibits Proliferation in HT29 ColonCancer Cells.

FIG. 95. Knockdown of CD49f mRNA Inhibits Proliferation in HCT116 ColonCancer Cells.

FIG. 96. Knockdown of CD49f mRNA Inhibits Proliferation in NCI-N87Gastric Cancer Cells.

FIG. 97. Anti-CD49f Antibody blocked H1299 Lung Tumor Cell LineProliferation.

FIG. 98. mRNA Sequence of CD49f, indicating siRNA target regions.

CD98

FIG. 99. CD98 is Over-expressed in Multiple Tumor Types.

FIG. 100. CD98 is Over-expressed in Colon Tumors.

FIG. 101. CD98 is Over-expression in Colorectal Tumors by QFACS.

FIG. 102. CD98 is Over-expressed in Colon Tumors Relative to NormalColon and CD45+ Cells.

FIG. 103. CD98 Expression in PBMC and Bone Marrow by FACS.

FIG. 104. CD980-ver-expression in Lung Tumors by QFACS.

FIG. 105. CD98 mRNA Expression Analysis in Multiple Tumor Tissues.

FIG. 106. Knockdown of CD98 mRNA Inhibits Proliferation in Pancreas,Lung and Breast Cancer Cells.

FIG. 107. Knockdown of CD98 mRNA Inhibits Proliferation in H1299 LungCancer Cells.

FIG. 108. Knockdown of Alternative CD98 Subunit mRNA InhibitsProliferation in H1299 Lung Cancer Cells.

FIG. 109. Knockdown of CD98 mRNA Inhibits Proliferation in MPANC-96Pancreatic Cancer Cells.

FIG. 110. mRNA sequence for CD98 light chain (SLC7A5), indicating siRNAtarget regions.

CD104

FIG. 111. CD104 is Over-expressed in Multiple Tumor Types.

FIG. 112. CD104 is Over-expressed in Multiple Tumor Types.

FIG. 113. CD104 is Over-expressed in Colon Tissues as Measured by QFACS.

FIG. 114. CD104 mRNA Expression in Multiple Tumor Types.

FIG. 115. Knockdown of CD104 mRNA Inhibits Proliferation in Colon andBreast Cancer Cells.

FIG. 116. Knockdown of CD104 mRNA Inhibits Proliferation in HCT116 ColonCancer Cells.

FIG. 117. mRNA sequence of B4 Integrin (CD 104), indicating siRNA targetregions.

DPEP1

FIG. 118. DPEP1 is Over-Expressed in Colon Carcinoma.

FIG. 119. FACS Confirms Over-Expression of DPEP1 on the Surface of ColonTumors.

FIG. 120. DPEP1 mRNA Expression Analysis in Colon and Lung TumorTissues.

FIG. 121. Knockdown of DPEP1 mRNA Inhibits Proliferation in Colon CancerCells.

FIG. 122. Knockdown of DPEP1 mRNA Increases Apoptosis in Colon CancerCells.

FIG. 123. Knockdown of DPEP1 mRNA Inhibits Proliferation and InducesApoptosis in HCT116 Colon Cancer Cells.

FIG. 124. Knockdown of DPEP1 mRNA Inhibits Proliferation in HT29 ColonCancer Cells.

FIG. 125. Monoclonal Antibody to DPEP1 Inhibits Proliferation in ColonCancer Cells.

FIG. 126. DPEP1 Expression in Colon Cell Lines.

FIG. 127. mRNA sequence of DPEP1, indicating siRNA target regions.

Tissue Factor (TF)

FIG. 128. TF is Overexpressed in Multiple Tumor Types.

FIG. 129. TF Expression in PBMC and Bone Marrow.

FIG. 130. TF Expression in Hematopoietic Cells and Pancreatic Cell LinesMeasured by QFACS.

FIG. 131. TF mRNA Expression Analysis in Pancreatic and Lung TumorTissues.

FIG. 132. Knockdown of TF mRNA Inhibits Proliferation in Multiple CancerCells.

FIG. 133. RNAi Knockdown of TF mRNA Inhibits Proliferation in H1299 LungCancer Cells.

FIG. 134. RNAi Knockdown of TF mRNA Inhibits Proliferation in ASPC-1Pancreatic Cancer Cells.

FIG. 135. TF-Ligand Activates AKT Signaling Pathway.

FIG. 136. Elevated Expression of Tissue Factor mRNA in Pancreatic TumorTissues.

FIG. 137. QFACS validation of TF cell-surface tumor expression.

FIG. 138. mRNA sequence of Tissue Factor, indicating siRNA targetregions.

Na—K ATPase beta3

FIG. 139. Na—K ATPase β3 is Overexpressed in Multiple Tumor Types.

FIG. 140. Na—K ATPase β3 mRNA Overexpression Lung and Pancreas TumorPanel.

FIG. 141. RNAi Knockdown of Na—K ATPase β3 mRNA Inhibits Proliferationin Pancreatic, Lung, Colon, and Kidney Cancer Cells.

FIG. 142. RNAi Knockdown of Na—K ATPase β3 mRNA Induces Apoptosis inPancreatic, Lung, Breast, Kidney and Gastric Cancer Cells.

FIG. 143. RNAi Knockdown of Na—K ATPase β3 mRNA Inhibit Proliferationand Induces Apoptosis in MPANC-96 Pancreatic Cancer Cells.

FIG. 144. RNAi Knockdown of Na—K ATPase β3 mRNA Inhibit Proliferationand Induces Apoptosis in ASPC-1 Pancreatic Cancer Cells.

FIG. 145. RNAi Knockdown of Na—K ATPase β3 mRNA Inhibit Proliferationand Induces Apoptosis in Caki-1 Kidney Cancer Cells.

FIG. 146. Anti-Na—K ATPase β3 Antibody Inhibits Proliferation of ASPC-1and BXPC-3 Pancreatic Cancer Cells.

FIG. 147. Na—K ATPase β3 siRNA in Combination with Gemzar IncreasesApoptosis of BXPC-3 Pancreatic Cancer Cells.

FIG. 148. mRNA sequence of Na/K ATPase beta 3, indicating siRNA targetregions.

VIPR1

FIG. 149. VIPR1 is Over-expressed in Multiple Tumor Types.

FIG. 150. VIPR1 mRNA Expression in Breast and Ovarian Tumors and NormalTissues.

FIG. 151. VIPR1 siRNA Screen Data—Anti-Proliferation Activity.

FIG. 152. VIPR1 Individual siRNA Duplex Data—Anti-ProliferationActivity—H1299 Lung Carcinoma.

FIG. 153. VIPR1 Individual siRNA Duplex Data—Anti-ProliferationActivity—Calu-1 Lung Carcinoma.

FIG. 154. VIPR1 (0186) Individual siRNA Duplex Data—Anti-ProliferationActivity—HCT116 Colon Carcinoma.

FIG. 155. Polyclonal Antibody to VIPR1 (04-0186) Inhibits Proliferationin Colon and Breast Cancer Cells.

FIG. 156. mRNA sequence of VIPR1, indicating siRNA target regions.

CD26

FIG. 157. CD26 is Over-Expressed in Multiple Tumor Types.

FIG. 158. CD26 mRNA Expression Analysis in Lung and Colon Tumor Tissues.

FIG. 159. CD26 QFACS Over-expression in Lung and Colon Tumors.

FIG. 160. CD26 QFACS Over-expression in Lung and Colon Tumors.

FIG. 161. CD26 QFACS Reveals Low Expression in Blood and Bone Marrow.

FIG. 162. Knockdown of CD26 mRNA Inhibits Proliferation in Lung,Gastric, Colon, Breast, Kidney, Gastric and Spheroid Cancer Cells.

FIG. 163. Knockdown of CD26 mRNA Induces Apoptosis in Spheroid CancerCells.

FIG. 164. Knockdown of CD26 mRNA Inhibits Proliferation of Calu-1 LungCancer Cells.

FIG. 165. Knockdown of CD26 Inhibits Proliferation in H1299 Lung CancerCells.

FIG. 166. Knockdown of CD26 Inhibits Proliferation in Caki-1 KidneyCancer Cells.

FIG. 167. Knockdown of CD26 Inhibits Proliferation in NCI-N87 GastricCancer Cells.

FIG. 168. Knockdown of CD26 Inhibits Proliferation and Induces Apoptosisin H1299-HES Spheroid Cancer Cells.

FIG. 169. Monoclonal Antibody to CD26 Inhibits Proliferation in Lung andColon Cancer Cells.

FIG. 170. mRNA sequence of CD26, indicating siRNA target regions.

CXADR

FIG. 171. CXADR is Overexpressed in Multiple Tumor Types.

FIG. 172. CXADR Expression by FACS in Colon and Lung Cancer Cell Lines.

FIG. 173. CXADR Expression by FACS in 3D Spheroid Cells Derived fromKidney and Lung Cancer Cell Lines.

FIG. 174. CXADR mRNA Expression Analysis in Multiple Tumor Tissues.

FIG. 175. Knockdown of CXADR mRNA Inhibits Proliferation in Lung, Colonand Gastric Cancer Cells.

FIG. 176. Knockdown of CXADR mRNA Inhibits Proliferation in HCT116 ColonCells.

FIG. 177. Knockdown of CXADR mRNA Inhibits Proliferation in HT29 ColonCells.

FIG. 178. Monoclonal Antibody to CXADR Inhibits Proliferation in Colonand Lung Cancer Cells.

FIG. 179. CXADR mRNA is Overexpressed in Colon Tumor Tissues.

FIG. 180. CXADR is Highly Expressed at mRNA Level in Cancer Cell Lines.

FIG. 181. CXADR Expression by FACS in 3D Spheroid Cells Derived fromColon and Lung Cancer Cell Lines.

FIG. 182. mRNA sequence of CXADR, indicating siRNA target regions.

PTK7

FIG. 183. PTK7 is Over-Expressed in Multiple Tumor Types.

FIG. 184. PTK7 is Expressed in Hormone-Dependent and Hormone-IndependentProstate Xenografts.

FIG. 185. PTK7 Is Expressed in H1299 Lung Cell Line.

FIG. 186. PTK7 Expression Observed on 3D Tumor Spheroid Cells.

FIG. 187. PTK7 mRNA Expression Analysis in Multiple Tumor Tissues.

FIG. 188. Knockdown of PTK7 (02-0262) mRNA Inhibits Proliferation inMultiple Cell Lines.

FIG. 189. Knockdown of PTK7 (02-0262) mRNA Induces Apoptosis in MultipleCancer Cell Lines.

FIG. 190. Knockdown of PTK7 (02-0262) mRNA Inhibits Proliferation andInduces Apoptosis in H1299 Lung Cancer Cells.

FIG. 191. Knockdown of PTK7 (02-0262) mRNA Inhibits Proliferation inHCT116 Colon Cancer Cells.

FIG. 192. Anti-PTK7 (02-0262) Ab Blocked H1299 Lung Tumor Cell LineProliferation.

FIG. 193. PTK7 Copy Number Increase by CGH.

FIG. 194. PTK7 Expression on 3D Spheroid Cells Derived from ACHN KidneyCancer Cell Line.

FIG. 195. PTK7 Expression in LnCAP Xenograft Isolated Cells.

FIG. 196. PTK7 is Expressed on Prostate Cell Lines as Measured by FlowCytometry.

FIG. 197. PTK7 is Expressed by FACS on Prostate Cell Lines.

FIG. 198. PTK7 is Expressed on Prostate Cell Lines as Measured by FlowCytometry.

FIG. 199. PTK7 is Expressed on Prostate Cell Lines as Measured by FlowCytometry.

FIG. 200. PTK7 is Expressed on Prostate Cell Lines as Measured by FlowCytometry.

FIG. 201. PTK7 is Expressed on Prostate Cell Lines as Measured by FlowCytometry.

FIG. 202. PTK7 mRNA Expression in Prostate Tumors and Normal TissuesDemonstrates Two Populations in Prostate Tumors.

FIG. 203. PTK7 Expression in 3D Tumor Spheroid Cells Derived from Kidneyand Lung Cancer Cell Lines.

FIG. 204. Copy Number Increase by CGH.

FIG. 205. mRNA sequence of PTK7, indicating siRNA target regions.

MISTR

FIG. 206. MISTR is Over-Expressed in Multiple Tumor Types.

FIG. 207. MISTR mRNA Expression Analysis in Multiple Tumor Tissues.

FIG. 208. Knockdown of MISTR mRNA Inhibits Proliferation in Pancreas,Lung, Colon, Kidney and Gastric Cancer Cells.

FIG. 209. Knockdown of MISTR mRNA Induces Apoptosis in Pancreas, Lungand Colon Cancer Cells.

FIG. 210. Knockdown of MISTR mRNA Inhibits Proliferation in HCT116 ColonCancer Cells.

FIG. 211. Knockdown of MISTR mRNA Inhibits Proliferation in MPANC96Pancreatic Cancer Cells.

FIG. 212. Anti-MISTR Polyclonal Antibody Inhibits Proliferation in LungCancer Cells.

FIG. 213. mRNA sequence of MISTR, indicating siRNA target regions.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. CAT Proteins andPeptides

The present invention provides the following targets and methods ofusing these targets: GFRa1, Claudin-4, ASCT2, CD166-ALCAM, CD55, TG2,CD49f, CD98, CD104, DPEP1, Tissue Factor (TF), Na—K ATPase beta3, VIPR1,CD26, CXADR, PTK7, and MISTR (see Figures), which are collectivelyreferred to herein as “CAT” (cancer-associated targets). In particular,the present invention provides methods of using these targets fordiagnosing and treating cancer. Each of these targets is associated withspecific types of cancers in particular, as shown in the Figures anddescribed in section 14 of the Examples section (“Summary ofexperimental validation”).

The present invention provides isolated CAT peptides and proteinsconsisting of, consisting essentially of, or comprising the amino acidsequences of SEQ ID NOS:1-4, 9, 11-12, 16-19, 24-36, 51-59, 69-74,81-85, 91-102, 115-116, 119-121, 127-134, 144-145, 148-149, 152-159,168-174, and 182-183, respectively encoded by the nucleic acid moleculeshaving the nucleotide sequences of SEQ ID NOS:5-8, 10, 13-15, 20-23,37-50, 60-68, 75-80, 86-90, 103-114, 117-118, 122-126, 135-143, 146-147,150-151, 160-167, 175-181, 184-185, as well as all obvious variants ofthese peptides that are within the art to make and use. Some of thesevariants are described in detail below.

A CAT peptide or protein can be attached to heterologous sequences toform chimeric or fusion proteins. Such chimeric and fusion proteinscomprise a peptide operatively linked to a heterologous protein havingan amino acid sequence not substantially homologous to the peptide.“Operatively linked” indicates that the peptide and the heterologousprotein are fused in-frame. The heterologous protein can be fused to theN-terminus or C-terminus of the peptide.

In some uses, the fusion protein does not affect the activity of thepeptide or protein per se. For example, the fusion protein can include,but is not limited to, fusion proteins, for example beta-galactosidasefusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged,HI-tagged and Ig fusions. Such fusion proteins, particularly poly-H isfusions, can facilitate the purification of recombinant CAT proteins orpeptides. In certain host cells (e.g., mammalian host cells), expressionand/or secretion of a protein can be increased by using a heterologoussignal sequence.

A chimeric or fusion CAT protein or peptide can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A CAT-encoding nucleic acid can becloned into such an expression vector such that the fusion moiety islinked in-frame to the CAT protein or peptide.

Variants of a CAT protein can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the CAT peptides of the presentinvention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package, usingeither a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16,14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Inyet another preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (Devereux, J., et al., Nucleic Acids Res. 12(1):387(1984)), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In anotherembodiment, the percent identity between two amino acid or nucleotidesequences is determined using the algorithm of E. Myers and W. Miller(CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4.

The nucleic acids and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Allelic variants of a CAT peptide can readily be identified as being ahuman protein having a high degree (significant) of sequencehomology/identity to at least a portion of the CAT peptide as well asbeing encoded by the same genetic locus as the CAT peptide providedherein. Genetic locus can readily be determined based on the genomicinformation. As used herein, two proteins (or a region of the proteins)have significant homology when the amino acid sequences are typically atleast about 70-80%, 80-90%, and more typically at least about 90-95% ormore homologous. A significantly homologous amino acid sequence,according to the present invention, will be encoded by a nucleic acidsequence that will hybridize to a CAT peptide encoding nucleic acidmolecule under stringent conditions as more fully described below.

Paralogs of a CAT peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the CAT peptide, as being encoded by a gene from humans, and ashaving similar activity or function. Two proteins will typically beconsidered paralogs when the amino acid sequences are typically at leastabout 60% or greater, and more typically at least about 70% or greaterhomology through a given region or domain. Such paralogs will be encodedby a nucleic acid sequence that will hybridize to a CAT peptide encodingnucleic acid molecule under moderate to stringent conditions as morefully described below.

Orthologs of a CAT peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the CAT peptide as well as being encoded by a gene from anotherorganism. Preferred orthologs will be isolated from mammals, preferablyprimates, for the development of human therapeutic targets and agents.Such orthologs will be encoded by a nucleic acid sequence that willhybridize to a CAT peptide-encoding nucleic acid molecule under moderateto stringent conditions, as more fully described below, depending on thedegree of relatedness of the two organisms yielding the proteins.

Non-naturally occurring variants of the CAT peptides of the presentinvention can readily be generated using recombinant techniques. Suchvariants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the CAT peptide. Forexample, one class of substitutions is conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a CAT peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

Variant CAT peptides can be fully functional or can lack function in oneor more activities, e.g. ability to bind substrate, ability tophosphorylate substrate, ability to mediate signaling, etc. Fullyfunctional variants typically contain only conservative variation orvariation in non-critical residues or in non-critical regions.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity such as CAT activity or in assays such as an invitro proliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

The present invention further provides fragments of CAT, in addition toand peptides that comprise and consist of such fragments. As usedherein, a fragment comprises at least 8, 10, 12, 14, 16, 18, 20 or morecontiguous amino acid residues of a CAT protein. Such fragments can bechosen based on the ability to retain one or more of the biologicalactivities of a CAT or could be chosen for the ability to perform afunction, e.g. bind a substrate or act as an immunogen. Particularlyimportant fragments are biologically active fragments, peptides thatare, for example, about 8 or more amino acids in length. Such fragmentswill typically comprise a domain or motif of a CAT, e.g., active site, atransmembrane domain or a substrate-binding domain. Further, possiblefragments include, but are not limited to, domain or motif containingfragments, soluble peptide fragments, and fragments containingimmunogenic structures. Predicted domains and functional sites arereadily identifiable by computer programs well known and readilyavailable to those of skill in the art (e.g., PROSITE analysis).

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in CAT are described inbasic texts, detailed monographs, and the research literature, and theyare well known to those of skill in the art.

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

Accordingly, CAT proteins of the present invention also encompassderivatives or analogs in which a substituted amino acid residue is notone encoded by the genetic code, in which a substituent group isincluded, in which a mature CAT is fused with another compound, such asa compound to increase the half-life of a CAT (for example, polyethyleneglycol), or in which the additional amino acids are fused to a matureCAT, such as a leader or secretory sequence or a sequence forpurification of a mature CAT or a pro-protein sequence.

2. Antibodies Against CAT Protein or Fragments Thereof

Antibodies that selectively bind to a CAT protein or peptides of thepresent invention can be made using standard procedures known to thoseof ordinary skills in the art. The term “antibody” is used in thebroadest sense, and specifically covers monoclonal antibodies (includingfull length monoclonal antibodies), polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), humanized antibody andantibody fragments (e.g., Fab, F(ab′).sub.2 and Fv) so long as theyexhibit the desired biological activity. Antibodies (Abs) andimmunoglobulins (Igs) are glycoproteins having the same structuralcharacteristics. While antibodies exhibit binding specificity to aspecific antigen, immunoglobulins include both antibodies and otherantibody-like molecules that lack antigen specificity.

As used herein, antibodies are usually heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two identical light (L) chains andtwo identical heavy (H) chains. Each light chain is linked to a heavychain by one covalent disulfide bond, while the number of disulfidelinkages varies between the heavy chains of different immunoglobulinisotypes. Each heavy and light chain also has regularly spacedintrachain disulfide bridges. Each heavy chain has at one end a variabledomain (VH) followed by a number of constant domains. Each light chainhas a variable domain at one end (VL) and a constant domain at its otherend. The constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light andheavy chain variable domains. Chothia et al., J. Mol. Biol. 186, 651-63(1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82 4592-4596(1985).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of the environment in which it isproduced. Contaminant components of its production environment arematerials that would interfere with diagnostic or therapeutic uses forthe antibody, and may include enzymes, hormones, and other proteinaceousor nonproteinaceous solutes. In preferred embodiments, the antibody willbe purified as measurable by at least three different methods: 1) togreater than 95% by weight of antibody as determined by the Lowrymethod, and most preferably more than 99% by weight; 2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator; or 3) tohomogeneity by SDS-PAGE under reducing or non-reducing conditions usingCoomasie blue or, preferably, silver stain. Isolated antibody includesthe antibody in situ within recombinant cells since at least onecomponent of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An “antigenic region” or “antigenic determinant” or an “epitope”includes any protein determinant capable of specific binding to anantibody. This is the site on an antigen to which each distinct antibodymolecule binds. Epitopic determinants usually consist of active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three-dimensional structural characteristics, aswell as charge characteristics.

“Antibody specificity,” is an antibody, which has a stronger bindingaffinity for an antigen from a first subject species than it has for ahomologue of that antigen from a second subject species. Normally, theantibody “bind specifically” to a human antigen (i.e., has a bindingaffinity (Kd) value of no more than about 1×10⁻⁷ M, preferably no morethan about 1×10⁻⁸ M and most preferably no more than about 1×10⁻⁹ M) buthas a binding affinity for a homologue of the antigen from a secondsubject species which is at least about 50 fold, or at least about 500fold, or at least about 1000 fold, weaker than its binding affinity forthe human antigen. The antibody can be of any of the various types ofantibodies as defined above, but preferably is a humanized or humanantibody (Queen et al., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762;and 6,180,370).

The present invention provides an “antibody variant,” which refers to anamino acid sequence variant of an antibody wherein one or more of theamino acid residues have been modified. Such variant necessarily haveless than 100% sequence identity or similarity with the amino acidsequence having at least 75% amino acid sequence identity or similaritywith the amino acid sequence of either the heavy or light chain variabledomain of the antibody, more preferably at least 80%, more preferably atleast 85%, more preferably at least 90%, and most preferably at least95%. Since the method of the invention applies equally to bothpolypeptides, antibodies and fragments thereof, these terms aresometimes employed interchangeably.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the antigen binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Papaindigestion of antibodies produces two identical antigen bindingfragments, called the Fab fragment, each with a single antigen bindingsite, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen binding fragments which are capable of crosslinkingantigen, and a residual other fragment (which is termed pFc′).Additional fragments can include diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments. As used herein, “functional fragment” withrespect to antibodies, refers to Fv, F(ab) and F(ab′)₂ fragments.

An “Fv” fragment is the minimum antibody fragment that contains acomplete antigen recognition and binding site. This region consists of adimer of one heavy and one light chain variable domain in a tight,non-covalent association (V_(H)-V_(L) dimer). It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six CDRs confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen, although at a lower affinity than the entirebinding site.

The Fab fragment [also designated as F(ab)] also contains the constantdomain of the light chain and the first constant domain (CH1) of theheavy chain. Fab′ fragments differ from Fab fragments by the addition ofa few residues at the carboxyl terminus of the heavy chain CH1 domainincluding one or more cysteines from the antibody hinge region. Fab′-SHis the designation herein for Fab′ in which the cysteine residue(s) ofthe constant domains have a free thiol group. F(ab′) fragments areproduced by cleavage of the disulfide bond at the hinge cysteines of theF(ab′)₂ pepsin digestion product. Additional chemical couplings ofantibody fragments are known to those of ordinary skill in the art.

The present invention further provides monoclonal antibody, polyclonalantibody as well as humanized antibody. In general, to generateantibodies, an isolated peptide is used as an immunogen and isadministered to a mammalian organism, such as a rat, rabbit or mouse.The full-length protein, an antigenic peptide fragment or a fusionprotein of a CAT protein can be used. Particularly important fragmentsare those covering functional domains. Many methods are known forgenerating and/or identifying antibodies to a given target peptide.Several such methods are described by Harlow, Antibodies, Cold SpringHarbor Press, (1989).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In additional to their specificity, the monoclonal antibodiesare advantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”antibody indicates the character of the antibody as being obtained froma substantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler and Milstein, Nature 256, 495 (1975), or may be madeby recombinant methods, e.g., as described in U.S. Pat. No. 4,816,567.The monoclonal antibodies for use with the present invention may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352: 624-628 (1991), as well as in Marks et al.,J. Mol. Biol. 222: 581-597 (1991). For detailed procedures for making amonoclonal antibody, see the Example below.

“Humanized” forms of non-human (e.g. murine or rabbit) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′).sub.2 or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibody may comprise residues, which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and optimizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see: Jones et al., Nature 321,522-525 (1986); Reichmann et al., Nature 332, 323-327 (1988) and Presta,Curr. Op. Struct. Biol. 2, 593-596 (1992).

Polyclonal antibodies may be prepared by any known method ormodifications of these methods including obtaining antibodies frompatients. For example, a complex of an immunogen such as a CAT protein,peptides or fragments thereof and a carrier protein is prepared and ananimal is immunized by the complex according to the same manner as thatdescribed with respect to the above monoclonal antibody preparation andthe description in the Example. A serum or plasma containing theantibody against the protein is recovered from the immunized animal andthe antibody is separated and purified. The gamma globulin fraction orthe IgG antibodies can be obtained, for example, by use of saturatedammonium sulfate or DEAE SEPHADEX, or other techniques known to thoseskilled in the art.

The antibody titer in the antiserum can be measured according to thesame manner as that described above with respect to the supernatant ofthe hybridoma culture. Separation and purification of the antibody canbe carried out according to the same separation and purification methodof antibody as that described with respect to the above monoclonalantibody and in the Example.

The protein used herein as the immunogen is not limited to anyparticular type of immunogen. In one aspect, antibodies are preferablyprepared from regions or discrete fragments of a CAT protein. Antibodiescan be prepared from any region of the proteins described herein. Inparticular, the proteins are selected from a group consisting of SEQ IDNOS:1-4, 9, 11-12, 16-19, 24-36, 51-59, 69-74, 81-85, 91-102, 115-116,119-121, 127-134, 144-145, 148-149, 152-159, 168-174, and 182-183 andfragments thereof. An antigenic fragment will typically comprise atleast 8 contiguous amino acid residues. The antigenic peptide cancomprise, however, at least 10, 12, 14, 16 or more amino acid residues.Such fragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness.

Antibodies may also be produced by inducing production in the lymphocytepopulation or by screening antibody libraries or panels of highlyspecific binding reagents as disclosed in Orlandi et al. (1989; ProcNatl Acad Sci 86:3833-3837) or Winter et al. (1991; Nature 349:293-299).A protein may be used in screening assays of phagemid or B-lymphocyteimmunoglobulin libraries to identify antibodies having a desiredspecificity. Numerous protocols for competitive binding or immunoassaysusing either polyclonal or monoclonal antibodies with establishedspecificities are well known in the art. Smith G. P., 1991, Curr. Opin.Biotechnol. 2: 668-673.

The antibodies of the present invention can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In a particular, such phage can be utilized to display antigen-bindingdomains expressed from a repertoire or combinatorial antibody library(e.g., human or murine). Phage expressing an antigen binding domain thatbinds the antigen of interest can be selected or identified withantigen, e.g., using labeled antigen or antigen bound or captured to asolid surface or bead. Phage used in these methods are typicallyfilamentous phage including fd and M13 binding domains expressed fromphage with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

Antibody can be also made recombinantly. When using recombinanttechniques, the antibody variant can be produced intracellularly, in theperiplasmic space, or directly secreted into the medium. If the antibodyvariant is produced intracellularly, as a first step, the particulatedebris, either host cells or lysed fragments, is removed, for example,by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies that aresecreted to the periplasmic space of E. coli. Briefly, cell paste isthawed in the presence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 minutes. Cell debriscan be removed by centrifugation. Where the antibody variant is secretedinto the medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore PELLICON ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibodies or antigen binding fragments may also be produced bygenetic engineering. The technology for expression of both heavy andlight chain genes in E. coli is the subject the following PCT patentapplications; publication number WO 901443, WO901443, and WO 9014424 andin Huse et al., 1989 Science 246:1275-1281. The general recombinantmethods are well known in the art.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique.

The suitability of protein A as an affinity ligand depends on thespecies and isotype of any immunoglobulin Fc domain that is present inthe antibody. Protein A can be used to purify antibodies that are basedon human .delta.1, .delta.2 or .delta.4 heavy chains (Lindmark et al.,J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for allmouse isotypes and for human .delta.3 (Guss et al., EMBO J. 5: 1567-1575(1986)). The matrix to which the affinity ligand is attached is mostoften agarose, but other matrices are available. Mechanically stablematrices such as controlled pore glass or poly(styrenedivinyl)benzeneallow for faster flow rates and shorter processing times than can beachieved with agarose. Where the antibody comprises a CH3 domain, theBAKERBOND ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful forpurification. Other techniques for protein purification such asfractionation on an ion-exchange column, ethanol precipitation, ReversePhase HPLC, chromatography on silica, chromatography on heparinSEPHAROSE chromatography on an anion or cation exchange resin (such as apolyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the antibody to berecovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

3. CAT Nucleic Acid Molecules

Isolated CAT nucleic acid molecules of the present invention consist of,consist essentially of, or comprise a nucleotide sequence that encodesCAT peptides of the present invention, an allelic variant thereof, or anortholog or paralog thereof. As used herein, an “isolated” nucleic acidmolecule is one that is separated from other nucleic acid present in thenatural source of the nucleic acid. Preferably, an “isolated” nucleicacid is free of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived.However, there can be some flanking nucleotide sequences, for example upto about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less, particularlycontiguous peptide encoding sequences and peptide encoding sequenceswithin the same gene but separated by introns in the genomic sequence.The important point is that the nucleic acid is isolated from remote andunimportant flanking sequences such that it can be subjected to thespecific manipulations described herein such as recombinant expression,preparation of probes and primers, and other uses specific to thenucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding a CAT peptide alone, the sequenceencoding the mature peptide and additional coding sequences, such as aleader or secretory sequence (e.g., a pre-pro or pro-protein sequence),the sequence encoding the mature peptide, with or without the additionalcoding sequences, plus additional non-coding sequences, for exampleintrons and non-coding 5′ and 3′ sequences such as transcribed butnon-translated sequences that play a role in transcription, mRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and stability of mRNA. In addition, the nucleic acid moleculemay be fused to a marker sequence encoding, for example, a peptide thatfacilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA obtained by cloningor produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the proteins of the present invention as well as nucleicacid molecules that encode obvious variants of a CAT protein of thepresent invention that are described above. Such nucleic acid moleculesmay be naturally occurring, such as allelic variants (same locus),paralogs (different locus), and orthologs (different organism), or maybe constructed by recombinant DNA methods or by chemical synthesis. Suchnon-naturally occurring variants may be made by mutagenesis techniques,including those applied to nucleic acid molecules, cells, or organisms.Accordingly, as discussed above, the variants can contain nucleotidesubstitutions, deletions, inversions and insertions. Variation can occurin either or both the coding and non-coding regions. The variations canproduce both conservative and non-conservative amino acid substitutions.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could at least 30, 40, 50, 100,250 or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encode epitopebearing regions of the peptide, or can be useful as DNA probes andprimers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence. Such nucleic acidmolecules can readily be identified as being able to hybridize undermoderate to stringent conditions, to the nucleotide sequence shown inthe Sequence Listing or a fragment of the sequence. Allelic variants canreadily be determined by genetic locus of the encoding gene.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions is hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

4. Vectors and Host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in prokaryotic or eukaryotic cellsor in both (shuttle vectors). Expression vectors contain cis-actingregulatory regions that are operably linked in the vector to the nucleicacid molecules such that transcription of the nucleic acid molecules isallowed in a host cell. The nucleic acid molecules can be introducedinto the host cell with a separate nucleic acid molecule capable ofaffecting transcription. Thus, the second nucleic acid molecule mayprovide a trans-acting factor interacting with the cis-regulatorycontrol region to allow transcription of the nucleic acid molecules fromthe vector. Alternatively, a trans-acting factor may be supplied by thehost cell. Finally, a trans-acting factor can be produced from thevector itself. It is understood, however, that in some embodiments,transcription and/or translation of the nucleic acid molecules can occurin a cell-free system.

The regulatory sequences to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(2001).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 3rd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (2001).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein; increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enteroenzyme. Typical fusion expression vectors includepGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorssuitable in a yeast host. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234(1987)), pMFa (Kurjan et al., Cell 30:933-943 (1982)), pJRY88 (Schultzet al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell. Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840 (1987)) and pMT2PC (Kaufman et al., EMBOJ. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance propagation or expression of the nucleic acidmolecules described herein. These are found for example in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(2001).

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 3rd. ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., (2001).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, which may be difficult toachieve with a multi-transmembrane domain-containing protein,appropriate secretion signals are incorporated into the vector. Thesignal sequence can be endogenous to the peptides or heterologous tothese peptides.

Where the peptide is not secreted into the medium, the protein can beisolated from the host cell by standard disruption procedures, includingfreeze thaw, sonication, mechanical disruption, use of lysing agents andthe like. The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing CATproteins or peptides that can be further purified to produce desiredamounts of CAT proteins or fragments. Thus, host cells containingexpression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involving aCAT protein or CAT protein fragments, such as those described above aswell as other formats known in the art. Thus, a recombinant host cellexpressing a native CAT protein is useful for assaying compounds thatstimulate or inhibit CAT protein function.

Host cells are also useful for identifying CAT protein mutants in whichthese functions are affected. If the mutants naturally occur and giverise to a pathology, host cells containing the mutations are useful toassay compounds that have a desired effect on the mutant CAT protein(for example, stimulating or inhibiting function) which may not beindicated by their effect on the native CAT protein.

5. Detection and Diagnosis in General

As used herein, a “biological sample” can be collected from tissues,blood, sera, cell lines or biological fluids such as, plasma,interstitial fluid, urine, cerebrospinal fluid, and the like, containingcells. In preferred embodiments, a biological sample comprises cells ortissues suspected of having diseases (e.g., cells obtained from abiopsy).

As used herein, a “differential level” is defined as the level of a CATprotein or nucleic acids in a test sample either above or below thelevel in control samples, wherein the level of control samples isobtained either from a control cell line, a normal tissue or bodyfluids, or combination thereof, from a healthy subject. While theprotein is overexpressed, the expression of a CAT is preferably greaterthan about 20%, or preferably greater than about 30%, and mostpreferably greater than about 50% or more of disease sample, at a levelthat is at least two fold, and preferably at least five fold, greaterthan the level of expression in control samples, as determined using arepresentative assay provided herein. While the protein is underexpressed, the expression of a CAT is preferably less than about 20%, orpreferably less than 30%, and most preferably less than about 50% ormore of the disease sample, at a level that is at least 0.5 fold, andpreferably at least 0.2 fold less than the level of the expression incontrol samples, as determined using a representative assay providedherein.

As used herein, a “subject” can be a mammalian subject or non mammaliansubject, preferably, a mammalian subject. A mammalian subject can behuman or non-human, preferably human. A healthy subject is defined as asubject without detectable diseases or associated pathologies by usingconventional diagnostic methods.

As used herein, the “disease(s)” preferably include cancer andassociated diseases and pathologies.

6. Treatment in General

This invention further pertains to novel agents identified by thescreening assays described below. It is also within the scope of thisinvention to use an agent identified for treatment purposes. Forexample, an agent identified as described herein (e.g., a CAT-modulatingagent, an antisense CAT nucleic acid molecule, a CAT-RNAi fragment, aCAT-specific antibody, or a CAT-binding partner) can be used in ananimal or other model to determine the efficacy, toxicity, or sideeffects of treatment with such an agent. Alternatively, an agentidentified as described herein can be used in an animal or other modelto determine the mechanism of action of such an agent. Furthermore, thisinvention pertains to uses of novel agents identified by theabove-described screening assays for treatments as described herein.

Modulators of CAT protein activity identified according to these drugscreening assays can be used to treat a subject with a disorder mediatedby a CAT, e.g., by treating cells or tissues that express a CAT at adifferential level. Methods of treatment include the steps ofadministering a modulator of CAT activity in a pharmaceuticalcomposition to a subject in need of such treatment.

The following terms, as used in the present specification and claims,are intended to have the meaning as defined below, unless indicatedotherwise.

“Treat,” “treating” or “treatment” of a disease includes: (1) inhibitingthe disease, i.e., arresting or reducing the development of the diseaseor its clinical symptoms, or (2) relieving the disease, i.e., causingregression of the disease or its clinical symptoms.

The term “prophylaxis” is used to distinguish from “treatment,” and toencompass both “preventing” and “suppressing,” it is not always possibleto distinguish between “preventing” and “suppressing,” as the ultimateinductive event or events may be unknown, latent, or the patient is notascertained until well after the occurrence of the event or events.Therefore, the term “protection,” as used herein, is meant to include“prophylaxis.”

A “therapeutically effective amount” means the amount of an agent that,when administered to a subject for treating a disease, is sufficient toeffect such treatment for the disease. The “therapeutically effectiveamount” will vary depending on the agent, the disease and its severityand the age, weight, etc., of the subject to be treated.

In one embodiment, when decreased expression or activity of the proteinis desired, an inhibitor, antagonist, antibody and the like or apharmaceutical agent containing one or more of these molecules may bedelivered. Such delivery may be effected by methods well known in theart and may include delivery by an antibody specifically targeted to theprotein.

In another embodiment, when increased expression or activity of theprotein is desired, the protein, an agonist, an enhancer and the like ora pharmaceutical agent containing one or more of these molecules may bedelivered. Such delivery may be effected by methods well known in theart.

While it is possible for the modulating agent to be administered in apure or substantially pure form, it is preferable to present it as apharmaceutical composition, formulation or preparation with a carrier.The formulations of the present invention, both for veterinary and forhuman use, comprise a suitable active CAT modulating agent, togetherwith one or more pharmaceutically acceptable carriers and, optionally,other therapeutic ingredients. The carrier(s) must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any method well-known in the pharmaceutical art.

Suitable pharmaceutical carriers include proteins such as albumins(e.g., U.S. Pat. No. 4,507,234, to Kato et al.), peptides andpolysaccharides such as aminodextran (e.g., U.S. Pat. No. 4,699,784, toShih et al.), or water. A carrier may also bear an agent by noncovalentbonding or by encapsulation, such as within a liposome vesicle (e.g.,U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriers specific forradionuclide agents include radiohalogenated small molecules andchelating compounds. For example, U.S. Pat. No. 4,735,792 disclosesrepresentative radiohalogenated small molecules and their synthesis. Aradionuclide chelate may be formed from chelating compounds that includethose containing nitrogen and sulfur atoms as the donor atoms forbinding the metal, metal oxide, radionuclide. For example, U.S. Pat. No.4,673,562, to Davison et al. discloses representative chelatingcompounds and their synthesis.

All methods include the step of bringing into association the activeingredient with the carrier, which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired formulation.

Formulations suitable for intravenous intramuscular, subcutaneous, orintraperitoneal administration conveniently comprise sterile aqueoussolutions of the active ingredient with solutions, which are preferablyisotonic with the blood of the recipient. Such formulations may beconveniently prepared by dissolving solid active ingredient in watercontaining physiologically compatible substances such as sodium chloride(e.g. 0.1-2.0M), glycine, and the like, and having a buffered pHcompatible with physiological conditions to produce an aqueous solution,and rendering said solution sterile. These may be present in unit ormulti-dose containers, for example, sealed ampoules or vials.

The formulations of the present invention may incorporate a stabilizer.Illustrative stabilizers are polyethylene glycol, proteins, saccharides,amino acids, inorganic acids, and organic acids, which may be usedeither on their own or as admixtures. These stabilizers are preferablyincorporated in an amount of 0.11-10,000 parts by weight per part byweight of immunogen. If two or more stabilizers are to be used, theirtotal amount is preferably within the range specified above. Thesestabilizers are used in aqueous solutions at the appropriateconcentration and pH. The specific osmotic pressure of such aqueoussolutions is generally in the range of 0.1-3.0 osmoles, preferably inthe range of 0.8-1.2. The pH of the aqueous solution is adjusted to bewithin the range of 5.0-9.0, preferably within the range of 6-8. Informulating the antibody of the present invention, anti-adsorption agentmay be used.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achievedthrough the use of polymer to complex or absorb the proteins or theirderivatives. The controlled delivery may be exercised by selectingappropriate macromolecules (for example polyester, polyamino acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled-release preparations is to incorporate an anti-CATantibody into particles of a polymeric material such as polyesters,polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetatecopolymers. Alternatively, instead of incorporating these agents intopolymeric particles, it is possible to entrap these materials inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly(methylmethacylate) microcapsules,respectively, or in colloidal drug delivery systems, for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules or in macroemulsions.

When oral preparations are desired, the compositions may be combinedwith typical carriers, such as lactose, sucrose, starch, talc magnesiumstearate, crystalline cellulose, methyl cellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic among others.

7. Diagnosis, Treatment and Screening Methods Using CAT Nucleic Acids

a. General Aspects

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as hybridization probes for messengerRNA, transcript/cDNA, and genomic DNA, such as to detect or isolatefull-length cDNA and genomic clones encoding CAT protein or peptide ofthe invention, or variants thereof.

The probes can correspond to any sequence along the entire length of thenucleic acid molecules of SEQ ID NOS:5-8, 10, 13-15, 20-23, 37-50,60-68, 75-80, 86-90, 103-114, 117-118, 122-126, 135-143, 146-147,150-151, 160-167, 175-181, 184-185. Accordingly, it could be derivedfrom 5′ noncoding regions, the coding region, and 3′ noncoding regions.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. The nucleic acid molecules arealso useful for expressing antigenic portions of the proteins.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

b. Diagnosis Methods

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. The probes can be used to detect the presence of, or todetermine levels of, a specific nucleic acid molecule in cells, tissues,and in organisms. Accordingly, probes corresponding to the peptidesdescribed herein can be used to assess expression and/or gene copynumber in a given cell, tissue, or organism. These uses are relevant fordiagnosis of disorders involving an increase or decrease in CAT proteinexpression relative to normal results.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express CAT protein differentially, such as bymeasuring a level of a CAT-encoding nucleic acid in a sample of cellsfrom a subject e.g., mRNA or genomic DNA, or determining if a CAT genehas been mutated.

The invention also encompasses kits for detecting the presence of CATnucleic acid in a biological sample. For example, the kit can comprisereagents such as a labeled or labelable nucleic acid or agent capable ofdetecting CAT nucleic acid in a biological sample; means for determiningthe amount of CAT nucleic acid in the sample; and means for comparingthe amount of CAT nucleic acid in the sample with a standard. Thecompound or agent can be packaged in a suitable container. The kit canfurther comprise instructions for using the kit to detect CAT proteinmRNA or DNA.

c. Screening Method Using Nucleic Acids

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate CAT nucleic acid expression.

The invention thus provides a method for identifying a compound that canbe used to treat a disease associated with differential expression of aCAT gene, particularly cancer. The method typically includes assayingthe ability of the compound to modulate the expression of CAT nucleicacid and thus identifying a compound that can be used to treat adisorder characterized by undesired CAT nucleic acid expression. Theassays can be performed in cell-based and cell-free systems. Cell-basedassays include cells naturally expressing CAT nucleic acid orrecombinant cells genetically engineered to express specific nucleicacid sequences.

The assay for CAT nucleic acid expression can involve direct assay ofnucleic acid levels, such as mRNA levels, or on collateral compoundsinvolved in the signal pathway. Further, the expression of genes thatare up- or down-regulated in response to a CAT protein signal pathwaycan also be assayed. In this embodiment the regulatory regions of thesegenes can be operably linked to a reporter gene such as luciferase.

Thus, modulators of CAT gene expression can be identified in a methodwherein a cell is contacted with a candidate compound or agent and theexpression of mRNA determined. The level of expression of CAT mRNA inthe presence of the candidate compound or agent is compared to the levelof expression of CAT mRNA in the absence of the candidate compound oragent. The candidate compound can then be identified as a modulator ofnucleic acid expression based on this comparison and be used, forexample to treat a disorder characterized by aberrant nucleic acidexpression. When expression of mRNA is statistically significantlygreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of nucleic acidexpression. When nucleic acid expression is statistically significantlyless in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of nucleic acidexpression.

d. Methods of Monitoring Treatment

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds or agents on the expression oractivity of a CAT gene in clinical trials or in a treatment regimen.Thus, the gene expression pattern can serve as a barometer for thecontinuing effectiveness of treatment with the compound, particularlywith compounds to which a patient can develop resistance. The geneexpression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

e. Treatment Using Nucleic Acid

The nucleic acid molecules are useful to design antisense constructs tocontrol CAT gene expression in cells, tissues, and organisms. A DNAantisense nucleic acid molecule is designed to be complementary to aregion of the gene involved in transcription, preventing transcriptionand hence production of CAT protein. An antisense RNA or DNA nucleicacid molecule would hybridize to the mRNA and thus block translation ofmRNA into CAT protein.

The nucleic acid of the present invention may also be used tospecifically suppress gene expression by methods such as RNAinterference (RNAi), which may also include cosuppression and quelling.This and antisense RNA or DNA of gene suppression are well known in theart. A review of this technique is found in Science 288:1370-1372, 2000.RNAi also operates on a post-transcriptional level and is sequencespecific, but suppresses gene expression far more efficiently thanantisense RNA. RNAi fragments, particularly double-stranded (ds) RNAi,can be also used to generate loss-of-function phenotypes.

The present invention relates to isolated RNA molecules(double-stranded; single-stranded) of from about 21 to about 25nucleotides which mediate RNAi. As used herein, about 21 to about 25 ntincludes nucleotides 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and29 nucleotides in length. The isolated RNAs of the present inventionmediate degradation of mRNA, the transcriptional product of a gene. SuchmRNA is also referred to herein as mRNA to be degraded. As used herein,the terms RNA, RNA molecule(s), RNA segment(s) and RNA fragment(s) areused interchangeably to refer to RNA that mediates RNA interference.These terms include double-stranded RNA, single-stranded RNA, isolatedRNA (partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA), as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of the 21-25nt RNA or internally (at one or more nucleotides of the RNA).Nucleotides in the RNA molecules of the present invention can alsocomprise non-standard nucleotides, including non-naturally occurringnucleotides or deoxyribonucleotides. Collectively, all such altered RNAsare referred to as analogs or analogs of naturally-occurring RNA. RNA of21-25 nucleotides of the present invention need only be sufficientlysimilar to natural RNA that it has the ability to mediate RNAi. As usedherein the phrase “mediates RNAi” refers to the ability to distinguishwhich RNAs are to be degraded by the RNAi machinery or process. RNA thatmediates RNAi interacts with the RNAi machinery such that it directs thedegradation of particular mRNAs. Such RNA may include RNAs of variousstructure, including short hairpin RNA.

In one embodiment, the present invention relates to RNA molecules ofabout 21 to about 25 nucleotides that direct cleavage of specific mRNAto which their sequence corresponds. It is not necessary that there beperfect correspondence of the sequences, but the correspondence must besufficient to enable the RNA to direct RNAi cleavage of the target mRNA(Holen et al. (2005) Nucleic Acids Res. 33, 4704-4710). In a particularembodiment, the 21-25 nt RNA molecules of the present invention comprisea 3′ hydroxyl group.

The present invention relates to 21-25 nt RNAs of specific genes,produced by chemical synthesis or recombinant DNA techniques, thatmediate RNAi. As used herein, the term isolated RNA includes RNAobtained by any means, including processing or cleavage of dsRNA;production by chemical synthetic methods; and production by recombinantDNA techniques. The invention further relates to uses of the 21-25 ntRNAs, such as for therapeutic or prophylactic treatment and compositionscomprising 21-25 nt RNAs that mediate RNAi, such as pharmaceuticalcompositions comprising 21-25 nt RNAs and an appropriate carrier.

The present invention also relates to a method of mediating RNAinterference of genes of a patient. In one embodiment, RNA of about 21to about 25 nt which targets the specific mRNA to be degraded isintroduced into a patient's cells. The cells are maintained underconditions allowing degradation of the mRNA, resulting in RNA-mediatedinterference of the mRNA of the gene in the cells of the patient.Treatment of patients with cancer with the RNAi will inhibit the growthand spread of the cancer and reduce the tumor. Treatment of patientsusing RNAi can also be in combination with other anti-cancer compounds.The RNAi may be used in combination with other treatment modalities,such as chemotherapy, cryotherapy, hyperthermia, radiation therapy, andother similar treatments. In one embodiment, a chemotherapy agent wascombined with the RNAi. In another embodiment, a chemotherapy namedGemzar was used.

Treatment of cancer or tumors in patients requires introduction of theRNA into the cancer or tumor cells. RNA may be directly introduced intothe cell, or introduced extracellularly into a cavity, interstitialspace, into the circulation of a patient, or introduced orally. Methodsfor oral introduction include direct mixing of the RNA with food, aswell as engineered approaches in which a species that is used as food isengineered to express the RNA and then ingested. Physical methods ofintroducing nucleic acids, for example, injection directly into the cellor extracellular injection into the patient, may also be used. Vascularor extravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the RNA may be introduced. RNA maybe introduced into an embryonic stem cell, or another multipotent cellderived from the patient. Physical methods of introducing nucleic acidsinclude injection of a solution containing the RNA, bombardment byparticles covered by the RNA, soaking cells or tissue in a solution ofthe RNA, or electroporation of cell membranes in the presence of theRNA. A viral construct packaged into a viral particle may be used tointroduce an expression construct into the cell, with the constructexpressing RNA. Other methods known in the art for introducing nucleicacids to cells may be used, such as lipid-mediated carrier transport,chemical-mediated transport, and the like. Thus the RNA may beintroduced along with components that perform one or more of thefollowing activities: enhance RNA uptake by the cell, promote annealingof the duplex strands, stabilize the annealed strands, or otherwiseincrease inhibition of the target gene. The RNAi may be used incombination with other treatment modalities, such as chemotherapy,cryotherapy, hyperthermia, radiation therapy, and the like.

The present invention may be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to tissue or patients. Preferredcomponents are the dsRNA and a vehicle that promotes introduction of thedsRNA. Such a kit may also include instructions to allow a user of thekit to practice the invention.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of CAT nucleic acid. Accordingly,these molecules can treat a disorder characterized by abnormal orundesired CAT nucleic acid expression. This technique involves cleavageby means of ribozymes containing nucleotide sequences complementary toone or more regions in the mRNA that attenuate the ability of the mRNAto be translated. Possible regions include coding regions andparticularly coding regions corresponding to the catalytic and otherfunctional activities of a CAT protein, such as substrate binding.

The nucleic acid molecules can be used for gene therapy in patientscontaining cells that are aberrant in CAT gene expression. Thus,recombinant cells, which include the patient's cells that have beenengineered ex vivo and returned to the patient, are introduced into anindividual where the cells produce a desired CAT protein to treat theindividual.

8. Diagnosis Using CAT Protein

Protein Detections

The present invention provides methods for diagnosing or detecting thedifferential presence of a CAT protein. Where a CAT is overexpressed indiseased cells, CAT protein can be detected directly.

The information obtained is also used to determine prognosis andappropriate course of treatment. For example, it is contemplated thatindividuals with a specific CAT expression or stage of disease mayrespond differently to a given treatment that individuals lacking CATexpression. The information obtained from the diagnostic methods of thepresent invention thus provides for the personalization of diagnosis andtreatment.

In one embodiment, the present invention provides a method formonitoring disease treatment in a subject comprising: determining thelevel of a CAT protein or any fragment(s) or peptide(s) thereof in atest sample from said subject, wherein a level of said CAT proteinsimilar to the level of said protein in a test sample from a healthysubject, or the level established for a healthy subject, is indicativeof successful treatment.

In another embodiment, the present invention provides a method fordiagnosing recurrence of disease following successful treatment in asubject comprising: determining the level of a CAT protein or anyfragment(s) or peptide(s) thereof in a test sample from said subject;wherein a changed level of said CAT protein relative to the level ofsaid protein in a test sample from a healthy subject, or the levelestablished for a healthy subject, is indicative of recurrence ofdiseases.

In yet another embodiment, the present invention provides a method fordiagnosing or detecting disease in a subject comprising: determining thelevel of a CAT protein or any fragment or peptides thereof in a testsample from said subject; wherein a differential level of said CATprotein relative to the level of said protein in a test sample from ahealthy subject, or the level established for a healthy subject, isindicative of disease.

These methods are also useful for diagnosing diseases that showdifferential protein expression. As describe earlier, normal, control orstandard values or level established from a healthy subject for proteinexpression are established by combining body fluids or tissue, cellextracts taken from a normal healthy mammalian or human subject withspecific antibodies to a protein under conditions for complex formation.Standard values for complex formation in normal and diseased tissues areestablished by various methods, often photometric means. Then complexformation as it is expressed in a subject sample is compared with thestandard values. Deviation from the normal standard and toward thediseased standard provides parameters for disease diagnosis or prognosiswhile deviation away from the diseased and toward the normal standardmay be used to evaluate treatment efficacy.

In yet another embodiment, the present invention provides a detection ordiagnostic method of a CAT by using LC/MS. The proteins from cells areprepared by methods known in the art (for example, R. Aebersold NatureBiotechnology, Volume 21, Number 6, June 2003). The differentialexpression of proteins in disease and healthy samples are quantitatedusing Mass Spectrometry and ICAT (Isotope Coded Affinity Tag) labeling,which is known in the art. ICAT is an isotope label technique thatallows for discrimination between two populations of proteins, such as ahealthy and a disease sample. The LC/MS spectra are collected for thelabeled samples. The raw scans from the LC/MS instrument are subjectedto peak detection and noise reduction software. Filtered peak lists arethen used to detect ‘features’ corresponding to specific peptides fromthe original sample(s). Features are characterized by their mass/charge,charge, retention time, isotope pattern and intensity.

The intensity of a peptide present in both healthy and disease samplescan be used to calculate the differential expression, or relativeabundance, of the peptide. The intensity of a peptide found exclusivelyin one sample can be used to calculate a theoretical expression ratiofor that peptide (singleton). Expression ratios are calculated for eachpeptide of each replicate of the experiment. Thus overexpression orunder expression of a CAT protein or peptide are similar to theexpression pattern in a test subject indicates the likelihood of havinga disease, particularly cancer, or an associated pathology.

Immunological methods for detecting and measuring complex formation as ameasure of protein expression using either specific polyclonal ormonoclonal antibodies are known in the art. Examples of such techniquesinclude enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays(RIAs), fluorescence-activated cell sorting (FACS) and antibody arrays.Such immunoassays typically involve the measurement of complex formationbetween the protein and its specific antibody. These assays and theirquantitation against purified, labeled standards are well known in theart (Ausubel, supra, unit 10.1-10.6). A two-site, monoclonal-basedimmunoassay utilizing antibodies reactive to two non-interferingepitopes is preferred, but a competitive binding assay may be employed(Pound (1998) Immunochemical Protocols, Humana Press, Totowa N.J.). Moreimmunological detections are described in section below.

For diagnostic applications, the antibody or its variant typically willbe labeled with a detectable moiety. Numerous labels are available whichcan be generally grouped into the following categories:

(a) Radioisotopes, such as ³⁶S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The antibodyvariant can be labeled with the radioisotope using the techniquesdescribed in Current Protocols in Immunology, vol 1-2, Coligen et al.,Ed., Wiley-Interscience, New York, Pubs. (1991) for example andradioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the antibody variant using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescence can be quantified using a fluorometer.

(c) Various enzyme-substrate labels are available and U.S. Pat. Nos.4,275,149 and 4,318,980 provide a review of some of these. The enzymegenerally catalyzes a chemical alteration of the chromogenic substratewhich can be measured using various techniques. For example, the enzymemay catalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for Use inEnzyme Immunoassay, in Methods in Enzyme. (Ed. J. Langone & H. VanVunakis), Academic press, New York, 73: 147-166 (1981).

Sometimes, the label is indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten (e.g. digoxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten antibody (e.g. anti-digoxin antibody). Thus,indirect conjugation of the label with the antibody can be achieved.

The biological samples can then be tested directly for the presence of aCAT by assays (e.g., ELISA or radioimmunoassay) and format (e.g.,microwells, dipstick, etc., as described in International PatentPublication WO 93/03367). Alternatively, proteins in the sample can besize separated (e.g., by polyacrylamide gel electrophoresis (PAGE)), inthe presence or absence of sodium dodecyl sulfate (SDS), and thepresence of CAT detected by immunoblotting (e.g., Western blotting).Immunoblotting techniques are generally more effective with antibodiesgenerated against a peptide corresponding to an epitope of a protein,and hence, are particularly suited to the present invention.

Antibody binding may be detected also by “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitation reactions,immunodiffusion assays, in situ immunoassays (e.g., using colloidalgold, enzyme or radioisotope labels, for example), precipitationreactions, agglutination assays (e.g., gel agglutination assays,hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many means are known in the art for detecting binding in animmunoassay and are within the scope of the present invention. As iswell known in the art, the immunogenic peptide should be provided freeof the carrier molecule used in any immunization protocol. For example,if the peptide is conjugated to KLH, it may be conjugated to BSA, orused directly, in a screening assay. In some embodiments, an automateddetection assay is utilized. Methods for the automation of immunoassaysare well known in the art (See e.g., U.S. Pat. Nos. 5,885,530,4,981,785, 6,159,750, and 5,358,691, each of which is hereinincorporated by reference). In some embodiments, the analysis andpresentation of results is also automated. For example, in someembodiments, software that generates a prognosis based on the presenceor absence of a series of antigens is utilized.

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample for binding with a limited amount ofantibody. The amount of antigen in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition. As a result, the standard and test sample thatare bound to the antibodies may conveniently be separated from thestandard and test sample, which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, or the proteinto be detected. In a sandwich assay, the test sample to be analyzed isbound by a first antibody, which is immobilized on a solid support, andthereafter a second antibody binds to the test sample, thus forming aninsoluble three-part complex. See e.g., U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S), so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or more CATtargets and the affinity value (Kd) is less than 1×10⁸ M.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art.

For immunohistochemistry, the disease tissue sample may be fresh orfrozen or may be embedded in paraffin and fixed with a preservative suchas formalin (see Example). The fixed or embedded section contains thesample are contacted with a labeled primary antibody and secondaryantibody, wherein the antibody is used to detect CAT protein expressionin situ. The detailed procedure is shown in the Example.

Antibodies against CAT proteins or peptides are useful to detect thepresence of one of the proteins of the present invention in cells ortissues to determine the pattern of expression of the protein amongvarious tissues in an organism and over the course of normaldevelopment.

Further, such antibodies can be used to detect protein in situ, invitro, or in a cell lysate or supernatant in order to evaluate theabundance and pattern of expression. Also, such antibodies can be usedto assess abnormal tissue distribution or abnormal expression duringdevelopment or progression of a biological condition. Antibody detectionof circulating fragments of the full length protein can be used toidentify turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. If a disorder is characterized by a specific mutation inthe protein, antibodies specific for this mutant protein can be used toassay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.The diagnostic uses can be applied, not only in genetic testing, butalso in monitoring a treatment modality. Accordingly, where treatment isultimately aimed at correcting expression level or the presence ofaberrant sequence and aberrant tissue distribution or developmentalexpression, antibodies directed against the protein or relevantfragments can be used to monitor therapeutic efficacy. More detectionand diagnostic methods are described in detail below.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools, as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Where a specificprotein has been correlated with expression in a specific tissue,antibodies that are specific for this protein can be used to identify atissue type.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array. Arrays are described in detail below fornucleic acid arrays and similar methods have been developed for antibodyarrays.

9. Methods of Treatment Based on CAT Proteins

a. Antibody Therapy

The antibody of the present invention can be used for therapeuticreasons. It is contemplated that the antibody of the present inventionmay be used to treat a mammal, preferably a human with a disease.

In general, the antibodies are also useful for inhibiting proteinfunction, for example, blocking the binding of a CAT protein or peptideto a binding partner such as a substrate. These uses can also be appliedin a therapeutic context in which treatment involves inhibiting theprotein's function. An antibody can be used, for example, to blockbinding, thus modulating (agonizing or antagonizing) the peptidesactivity. Antibodies can be prepared against specific fragmentscontaining sites required for function or against intact protein that isassociated within a cell or cell membrane. The functional blockingassays are provided in detail in the Examples.

The antibodies of present invention can also be used as means ofenhancing the immune response. The antibodies can be administered inamounts similar to those used for other therapeutic administrations ofantibody. For example, pooled gamma globulin is administered at a rangeof about 1 mg to about 100 mg per patient.

Antibodies reactive with CAT proteins or peptides can be administeredalone or in conjunction with other therapies, such as anti-cancertherapies, to a mammal afflicted with cancer or other disease. Examplesof anti-cancer therapies include, but are not limited to, chemotherapy,radiation therapy, and adoptive immunotherapy therapy with TIL (TumorInfiltration Lymphocytes).

The selection of an antibody subclass for therapy will depend upon thenature of the antigen to be acted upon. For example, an IgM may bepreferred in situations where the antigen is highly specific for thediseased target and rarely occurs on normal cells. However, where thedisease-associated antigen is also expressed in normal tissues, althoughat much lower levels, the IgG subclass may be preferred, since thebinding of at least two IgG molecules in close proximity is required toactivate complement, less complement mediated damage may occur in thenormal tissues which express smaller amounts of the antigen and,therefore, bind fewer IgG antibody molecules. Furthermore, IgG moleculesby being smaller may be more able than IgM molecules to localize to thediseased tissue.

The mechanism for antibody therapy is that the therapeutic antibodyrecognizes a cell surface protein or a cytosolic protein that isexpressed or preferably, overexpressed in a diseased cell. By NK cell orcomplement activation, or conjugation of the antibody with animmunotoxin or radiolabel, the interaction can abrogate ligand/receptorinteraction or activation of apoptosis.

The potential mechanisms of antibody-mediated cytotoxicity of diseasedcells are phagocyte (antibody dependent cellular cytotoxicity (ADCC))(see Example), complement (Complement-mediated cytotoxicity (CMC)) (seeExample), naked antibody (receptor cross-linking apoptosis and growthfactor inhibition), or targeted payload labeled with radionuclide orimmunotoxins or immunochemotherapeutics.

In one embodiment, the antibody is administered to a nonhuman mammal forthe purposes of obtaining preclinical data, for example. Exemplarynonhuman mammals to be treated include nonhuman primates, dogs, cats,rodents and other mammals in which preclinical studies are performed.Such mammals may be established animal models for a disease to betreated with the antibody or may be used to study toxicity of theantibody of interest. In each of these embodiments, dose escalationstudies may be performed on the mammal.

The antibody is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local immunosuppressive treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody variant issuitably administered by pulse infusion, particularly with decliningdoses of the antibody variant. Preferably the dosing is given byinjections, most preferably intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.

For the prevention or treatment of a disease, the appropriate dosage ofthe antibody will depend on the type of disease to be treated, theseverity and the course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician.

Depending on the type and severity of the disease, about 1 μg/kg to 150mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosagefor administration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment is sustaineduntil a desired suppression of disease symptoms occurs. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays.

The antibody composition will be formulated, dosed and administered in amanner consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

The therapeutically effective amount of the antibody to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat a disease or disorder. Theantibody may optionally be formulated with one or more agents currentlyused to prevent or treat the disorder in question.

Suitable agents in this regard include radionuclides, differentiationinducers, drugs, toxins, and derivatives thereof. Preferredradionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re ²¹¹At and ²¹²Bi. Preferred drugs include methotrexate, and pyrimidine and purineanalogs. Preferred differentiation inducers include phorbol esters andbutyric acid. Preferred toxins include ricin, abrin, diptheria toxin,cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, andpokeweed antiviral protein

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable antibody either directly or indirectly (e.g., via a linkergroup). A direct reaction between an agent and an antibody is possiblewhen each possesses a substituent capable of reacting with the other.For example, a nucleophilic group, such as an amino or sulfhydryl group,on one may be capable of reacting with a carbonyl-containing group, suchas an anhydride or an acid halide, or with an alkyl group containing agood leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be affected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g. U.S. Pat. No.4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways as described above.

b. Other Immunotherapy

Peptides derived from a CAT protein sequence may be modified to increasetheir immunogenicity by enhancing the binding of the peptide to the MHCmolecules in which the peptide is presented. The peptide or modifiedpeptide may be conjugated to a carrier molecule to enhance theantigenicity of the peptide. Examples of carrier molecules, include, butare not limited to, human albumin, bovine albumin, lipoprotein andkeyhole limpet hemo-cyanin (“Basic and Clinical Immunology” (1991)Stites, D. P. and Terr A. I. (eds) Appleton and Lange, Norwalk Conn.,San Mateo, Calif.).

An “immunogenic peptide” is a peptide, which comprises anallele-specific motif such that the peptide will bind the MHC allele(HLA in human) and be capable of inducing a CTL (cytotoxicT-lymphocytes) response. Thus, immunogenic peptides are capable ofbinding to an appropriate class I or II MHC molecule and inducing acytotoxic T cell or T helper cell response against the antigen fromwhich the immunogenic peptide is derived.

Alternatively, amino acid sequence variants of the peptide can beprepared by altering the nucleic acid sequence of the DNA which encodesthe peptide, or by peptide synthesis. At the genetic level, thesevariants ordinarily are prepared by site-directed mutagenesis ofnucleotides in the DNA encoding the peptide molecule, thereby producingDNA encoding the variant, and thereafter expressing the DNA inrecombinant cell culture. The variants typically exhibit the samequalitative biological activity as the nonvariant peptide.

Recombinant or natural CAT proteins, peptides, fragment thereof, ormodified peptides, may be used as a vaccine either prophylactically ortherapeutically. When provided prophylactically the vaccine is providedin advance of any evidence of disease, particularly, cancer. Theprophylactic administration of the disease vaccine should serve toprevent or attenuate diseases, preferably cancer, in a mammal.

Preparation of vaccine uses recombinant protein or peptide expressionvectors comprising a nucleic acid sequence encoding all or part of a CATprotein. Examples of vectors that may be used in the aforementionedvaccines include, but are not limited to, defective retroviral vectors,adenoviral vectors vaccinia viral vectors, fowl pox viral vectors, orother viral vectors (Mulligan, R. C., (1993) Science 260:926-932). Thevectors can be introduced into a mammal either prior to any evidence ofthe disease or to mediate regression of the disease in a mammalafflicted with disease. Examples of methods for administering the viralvector into the mammals include, but are not limited to, exposure ofcells to the virus ex vivo, or injection of the retrovirus or a producercell line of the virus into the affected tissue or intravenousadministration of the virus. Alternatively the vector may beadministered locally by direct injection into the cancer lesion ortopical application in a pharmaceutically acceptable carrier. Thequantity of viral vector, carrying all or part of a CAT nucleic acidsequence, to be administered is based on the titer of virus particles. Apreferred range may be about 10⁶ to about 10¹¹ virus particles permammal, preferably a human.

After immunization the efficacy of the vaccine can be assessed by theproduction of antibodies or immune cells that recognize the antigen, asassessed by specific lytic activity or specific cytokine production orby tumor regression. One skilled in the art would know the conventionalmethods to assess the aforementioned parameters. If the mammal to beimmunized is already afflicted with cancer, the vaccine can beadministered in conjunction with other therapeutic treatments. Examplesof other therapeutic treatments includes, but are not limited to,adoptive T cell immunotherapy, coadministration of cytokines or othertherapeutic drugs for cancer.

Alternatively, all or parts thereof of a substantially or partiallypurified CAT protein or peptides may be administered as a vaccine in apharmaceutically acceptable carrier. Ranges of the protein that may beadministered are about 0.001 to about 100 mg per patient, preferreddoses are about 0.01 to about 100 mg per patient. Immunization may berepeated as necessary, until a sufficient titer of anti-immunogenantibody or immune cells has been obtained.

In yet another alternative embodiment a viral vector, such as aretroviral vector, can be introduced into mammalian cells. Examples ofmammalian cells into which the retroviral vector can be introducedinclude, but are not limited to, primary mammalian cultures orcontinuous mammalian cultures, COS cells, NIH3T3, or 293 cells (ATTC#CRL 1573), dendritic cells. The means by which the vector carrying thegene may be introduced into a cell includes, but is not limited to,microinjection, electroporation, transfection or transfection using DEAEdextran, lipofection, calcium phosphate or other procedures known to oneskilled in the art (Sambrook et al. 3rd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (2001).

The vaccine formulation of the present invention comprises an immunogenthat induces an immune response directed against the cancer associatedantigen such as a CAT protein, and in nonhuman primates and finally inhumans. The safety of the immunization procedures is determined bylooking for the effect of immunization on the general health of theimmunized animal (weight change, fever, appetite behavior etc.) andlooking for pathological changes on autopsies. After initial testing inanimals, cancer patients can be tested. Conventional methods would beused to evaluate the immune response of the patient to determine theefficiency of the vaccine.

In one embodiment mammals, preferably human, at high risk for disease,particularly cancer, are prophylactically treated with the vaccines ofthis invention. Examples include, but are not limited to, humans with afamily history of a disease, humans with a history of disease,particular cancer, or humans afflicted with a disease, such as cancerthat has been previously resected and therefore at risk forreoccurrence. When provided therapeutically, the vaccine is provided toenhance the patient's own immune response to the disease antigen presenton the disease cells or present during advanced stage of the disease.The vaccine, which acts as an immunogen, may be a cell, cell lysate fromcells transfected with a recombinant expression vector, or a culturesupernatant containing the expressed protein. Alternatively, theimmunogen is a partially or substantially purified recombinant protein,peptide or analog thereof or modified peptides or analogs thereof. Theproteins or peptides may be conjugated with lipoprotein or administeredin liposomal form or with adjuvant.

While it is possible for the immunogen to be administered in a pure orsubstantially pure form, it is preferable to present it as apharmaceutical composition, formulation or preparation, as discussedhereinabove.

Vaccination can be conducted by conventional methods. For example, theimmunogen can be used in a suitable diluent such as saline or water, orcomplete or incomplete adjuvants. Further, the immunogen may or may notbe bound to a carrier to make the protein immunogenic. Examples of suchcarrier molecules include but are not limited to bovine serum albumin(BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid, and the like.The immunogen also may be coupled with lipoproteins or administered inliposomal form or with adjuvants. The immunogen can be administered byany route-appropriate for antibody production such as intravenous,intraperitoneal, intramuscular, subcutaneous, and the like. Theimmunogen may be administered once or at periodic intervals until asignificant titer of anti-CAT immune cells or anti-CAT antibody isproduced. The presence of anti-CAT immune cells may be assessed bymeasuring the frequency of precursor CTL (cytotoxic T-lymphocytes)against CAT antigen prior to and after immunization by a CTL precursoranalysis assay (Coulie, P. et al., (1992) International Journal OfCancer 50:289-297). The antibody may be detected in the serum using theimmunoassay described above.

The safety of the immunization procedures is determined by examining theeffect of immunization on the general health of the immunized animal(fever, change in weight, appetite, behavior etc.) and pathologicalchanges on autopsies. After initial testing in animals, human patientscan be tested. Conventional methods would be used to evaluate the immuneresponse of the patient to determine the efficiency of the vaccine.

In yet another embodiment of this invention, all or portions of a CATprotein or peptides or fragments thereof, or modified peptides, may beexposed to dendritic cells cultured in vitro. The cultured dendriticcells provide a means of producing T-cell dependent antigens comprisedof dendritic cell modified antigen or dendritic cells pulsed withantigen, in which the antigen is processed and expressed on the antigenactivated dendritic cell. The CAT antigen activated dendritic cells orprocessed dendritic cell antigens may be used as immunogens for vaccinesor for the treatment of diseases, particularly cancer. The dendriticcells should be exposed to the antigen for sufficient time to allow theantigens to be internalized and presented on the dendritic cellssurface. The resulting dendritic cells or the dendritic-cell processedantigens can then be administered to an individual in need of therapy.Such methods are described in Steinman et al. (WO93/208185) and inBanchereau et al. (EPO Application 0563485A1).

In yet another aspect of this invention T-cells isolated fromindividuals can be exposed to CAT proteins, peptides or fragmentthereof, or modified peptides in vitro and then administered to apatient in need of such treatment in a therapeutically effective amount.Examples of where T-lymphocytes can be isolated include but are notlimited to, peripheral blood cells lymphocytes (PBL), lymph nodes, ortumor infiltrating lymphocytes (TIL). Such lymphocytes can be isolatedfrom the individual to be treated or from a donor by methods known inthe art and cultured in vitro (Kawakami, Y. et al. (1989) J. Immunol.142: 2453-3461). Lymphocytes are cultured in media such as RPMI or RPMI1640 or AIM V for 1-10 weeks. Viability is assessed by trypan blue dyeexclusion assay. Examples of how these sensitized T-cells can beadministered to the mammal include but are not limited to,intravenously, intraperitoneally or intralesionally. Parameters that maybe assessed to determine the efficacy of these sensitized T-lymphocytesinclude, but are not limited to, production of immune cells in themammal being treated or tumor regression. Conventional methods are usedto assess these parameters. Such treatment can be given in conjunctionwith cytokines or gene modified cells (Rosenberg, S. A. et al. (1992)Human Gene Therapy, 3: 75-90; Rosenberg, S. A. et al. (1992) Human GeneTherapy, 3: 57-73).

The present invention is further described by the following examples,which are provided solely to illustrate the invention by reference tospecific embodiments. This exemplification, while illustrating certainaspects of the invention, does not offer the limitations or circumscribethe scope of the disclosed invention.

10. Screening Methods Using Proteins

CAT proteins can be used to identify compounds or agents that modulateactivity of a CAT protein in its natural state or an altered form thatcauses a specific disease or pathology associated with CAT. CAT of thepresent invention, as well as appropriate variants and fragments, can beused in high-throughput screens to assay candidate compounds for theability to bind to CAT. These compounds can be further screened againstfunctional CAT to determine the effect of the compound on CAT activity.Further, these compounds can be tested in animal or invertebrate systemsto determine activity/effectiveness. Compounds can be identified thatactivate (agonist) or inactivate (antagonist) CAT to a desired degree.

CAT of the present invention, as well as appropriate variants andfragments, can be used in high-throughput screening to assay candidatecompounds for the ability to bind to CAT. These compounds can be furtherscreened against functional CAT to determine the effect of the compoundon CAT activity. Further, these compounds can be tested in animal orinvertebrate systems to determine activity/effectiveness. Compounds canbe identified that activate (agonist) or inactivate (antagonist) CAT toa desired degree.

Further, the proteins of the present invention can be used to screen acompound or an agent for the ability to stimulate or inhibit interactionbetween a CAT protein and a molecule that normally interacts with theCAT protein, e.g. a substrate or an extracellular binding ligand or acomponent of the signal pathway that the CAT protein normally interacts(for example, a cytosolic signal protein). Such assays typically includethe steps of combining a CAT protein with a candidate compound underconditions that allow the CAT protein, or fragment, to interact with thetarget molecule, and to detect the formation of a complex between theprotein and the target or to detect the biochemical consequence of theinteraction with the CAT protein and the target, such as any of theassociated effects of signal transduction such as proteinphosphorylation, cAMP turnover, and adenylate cyclase activation, etc.

Candidate compounds or agents include 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)2, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound or agent is a soluble fragment of a CAT thatcompetes for substrate binding. Other candidate compounds include mutantCAT or appropriate fragments containing mutations that affect CATfunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

Any of the biological or biochemical functions mediated by a CAT can beused as an endpoint assay to identify an agent that modulates CATactivity. These include all of the biochemical or biochemical/biologicalevents described herein, in the references cited herein, incorporated byreference for these endpoint assay targets, and other functions known tothose of ordinary skill in the art or that can be readily identified.Specifically, a biological function of a cell or tissues that expressesCAT can be assayed.

A substrate-binding region can be used that interacts with a differentsubstrate than one which is recognized by a native CAT. Accordingly, adifferent set of signal transduction components is available as anend-point assay for activation. This allows for assays to be performedin other than the specific host cell from which a CAT is derived.

Competition binding assays may also be used to discover compounds thatinteract with a CAT (e.g. binding partners and/or ligands). Thus, acompound can be exposed to a CAT polypeptide under conditions that allowthe compound to bind or to otherwise interact with the polypeptide.Soluble CAT polypeptide is also added to the mixture. If the testcompound interacts with the soluble CAT polypeptide, it decreases theamount of complex formed or activity from CAT. This type of assay isparticularly useful in cases in which compounds are sought that interactwith specific regions of CAT. Thus, the soluble polypeptide thatcompetes with the target CAT region is designed to contain peptidesequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the CAT protein, or fragment, or its target moleculeto facilitate separation of complexes from uncomplexed forms of one orboth of the proteins, as well as to accommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione SEPHAROSE beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofCAT-binding protein found in the bead fraction quantitated from the gelusing standard electrophoretic techniques. For example, either thepolypeptide or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin using techniques well known inthe art. Alternatively, antibodies reactive with the protein but whichdo not interfere with binding of the protein to its target molecule canbe derivatized to the wells of the plate, and the protein trapped in thewells by antibody conjugation. Preparations of CAT-binding protein and acandidate compound are incubated in CAT protein-presenting wells and theamount of complex trapped in the well can be quantitated. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with a CAT protein target molecule, or which arereactive with CAT protein and compete with the target molecule, as wellas CAT-linked assays which rely on detecting an enzymatic activityassociated with the target molecule.

Agents that modulate a CAT of the present invention can be identifiedusing one or more of the above assays, alone or in combination. It isgenerally preferable to use a cell-based or cell free system first andthen confirm activity in an animal or other model system. Such modelsystems are well known in the art and can readily be employed in thiscontext.

In yet another aspect of the invention, a CAT protein can be used as a“bait protein” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with CAT and are involved in CAT activity. Such CAT-bindingproteins are also likely to be involved in the propagation of signals bya CAT protein or CAT targets as, for example, downstream elements of aCAT-mediated signaling pathway. Alternatively, such CAT-binding proteinsare likely to be CAT inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a CAT protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences that encode an unidentified protein (“prey” or“sample”) is fused to a gene that codes for the activation domain of theknown transcription factor. If the “bait” and the “prey” proteins areable to interact, in vivo, forming a CAT-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with the CATprotein.

Array:

“Array” refers to an ordered arrangement of at least two transcripts,proteins or peptides, or antibodies on a substrate. At least one of thetranscripts, proteins, or antibodies represents a control or standard,and the other transcript, protein, or antibody is of diagnostic ortherapeutic interest. The arrangement of at least two and up to about40,000 transcripts, proteins, or antibodies on the substrate assuresthat the size and signal intensity of each labeled complex, formedbetween each transcript and at least one nucleic acid, each protein andat least one ligand or antibody, or each antibody and at least oneprotein to which the antibody specifically binds, is individuallydistinguishable.

An “expression profile” is a representation of gene expression in asample. A nucleic acid expression profile is produced using sequencing,hybridization, or amplification technologies using transcripts from asample. A protein expression profile, although time delayed, minors thenucleic acid expression profile and is produced using gelelectrophoresis, mass spectrometry, or an array and labeling moieties orantibodies which specifically bind the protein. The nucleic acids,proteins, or antibodies specifically binding the protein may be used insolution or attached to a substrate, and their detection is based onmethods well known in the art.

A substrate includes but is not limited to, paper, nylon or other typeof membrane, filter, chip, glass slide, or any other suitable solidsupport.

The present invention also provides an antibody array. Antibody arrayshave allowed the development of techniques for high-throughput screeningof recombinant antibodies. Such methods use robots to pick and gridbacteria containing antibody genes, and a filter-based ELISA to screenand identify clones that express antibody fragments. Because liquidhandling is eliminated and the clones are arrayed from master stocks,the same antibodies can be spotted multiple times and screened againstmultiple antigens simultaneously. For more information, see de Wildt etal. (2000) Nat. Biotechnol. 18:989-94.

The array is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), U.S.Pat. No. 5,807,522, Brown et al., all of which are incorporated hereinin their entirety by reference.

In one embodiment, a nucleic acid array or a microarray, preferablycomposed of a large number of unique, single-stranded nucleic acidsequences, usually either synthetic antisense oligonucleotides orfragments of cDNAs, fixed to a solid support. The oligonucleotides arepreferably about 6-60 nucleotides in length, more preferably 15-30nucleotides in length, and most preferably about 20-25 nucleotides inlength.

In order to produce oligonucleotides to a known sequence for an array,the gene(s) of interest (or an ORF identified from the contigs of thepresent invention) is typically examined using a computer algorithmwhich starts at the 5′ or at the 3′ end of the nucleotide sequence.Typical algorithms will then identify oligomers of defined length thatare unique to the gene, have a GC content within a range suitable forhybridization, and lack predicted secondary structure that may interferewith hybridization. In certain situations it may be appropriate to usepairs of oligonucleotides on an array. The “pairs” will be identical,except for one nucleotide that preferably is located in the center ofthe sequence. The second oligonucleotide in the pair (mismatched by one)serves as a control. The number of oligonucleotide pairs may range fromtwo to one million. The oligomers are synthesized at designated areas ona substrate using a light-directed chemical process, wherein thesubstrate may be paper, nylon or other type of membrane, filter, chip,glass slide or any other suitable solid support as described above.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference.

A gene expression profile comprises the expression of a plurality oftranscripts as measured by after hybridization with a sample. Thetranscripts of the invention may be used as elements on an array toproduce a gene expression profile. In one embodiment, the array is usedto diagnose or monitor the progression of disease. Researchers canassess and catalog the differences in gene expression between healthyand diseased tissues or cells.

For example, the transcript or probe may be labeled by standard methodsand added to a biological sample from a patient under conditions for theformation of hybridization complexes. After an incubation period, thesample is washed and the amount of label (or signal) associated withhybridization complexes, is quantified and compared with a standardvalue. If complex formation in the patient sample is significantlyaltered (higher or lower) in comparison to either a normal or diseasestandard, then differential expression indicates the presence of adisorder.

In order to provide standards for establishing differential expression,normal and disease expression profiles are established. This isaccomplished by combining a sample taken from normal subjects, eitheranimal or human or nonmammal, with a transcript under conditions forhybridization to occur. Standard hybridization complexes may bequantified by comparing the values obtained using normal subjects withvalues from an experiment in which a known amount of a purified sequenceis used. Standard values obtained in this manner may be compared withvalues obtained from samples from patients who were diagnosed with aparticular condition, disease, or disorder. Deviation from standardvalues toward those associated with a particular disorder is used todiagnose that disorder.

By analyzing changes in patterns of gene expression, disease can bediagnosed at earlier stages before the patient is symptomatic. Theinvention can be used to formulate a prognosis and to design a treatmentregimen. The invention can also be used to monitor the efficacy oftreatment. For treatments with known side effects, the array is employedto improve the treatment regimen. A dosage is established that causes achange in genetic expression patterns indicative of successfultreatment. Expression patterns associated with the onset of undesirableside effects are avoided.

In another embodiment, animal models which mimic a human disease can beused to characterize expression profiles associated with a particularcondition, disease, or disorder; or treatment of the condition, disease,or disorder. Novel treatment regimens may be tested in these animalmodels using arrays to establish and then follow expression profilesover time. In addition, arrays may be used with cell cultures or tissuesremoved from animal models to rapidly screen large numbers of candidatedrug molecules, looking for ones that produce an expression profilesimilar to those of known therapeutic drugs, with the expectation thatmolecules with the same expression profile will likely have similartherapeutic effects. Thus, the invention provides the means to rapidlydetermine the molecular mode of action of a drug.

Such assays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies or in clinical trials orto monitor the treatment of an individual patient. Once the presence ofa condition is established and a treatment protocol is initiated,diagnostic assays may be repeated on a regular basis to determine if thelevel of expression in the patient begins to approximate that which isobserved in a normal subject. The results obtained from successiveassays may be used to show the efficacy of treatment over a periodranging from several days to years.

WORKING EXAMPLES 1. Tissue Processing and Cell Lines

Tissue Processing:

All tissues were procured as fresh specimens. Tissues were collected asremnant tissues following surgical resection of cancer tissues. Remnanttissues were supplied following processing for pathological diagnosisaccording to proper standards of patient care. Procurement of alltissues was performed in an anonymised manner in strict compliance withFederal mandated ethical and legal guidelines (HIPAA) and in accordancewith clinical institution ethical review board as well as the internalinstitutional review board. Tissues were transported on ice in ice-coldtransport buffer by courier for processing.

i) Enrichment of Epithelial Cells from Normal Tissue:

Normal tissue was transferred from the transport vessel to a steriledish containing 25 ml of ice-cold transport buffer. The tissue wasmeasured, weighed and photographed. The tissue was dissected to isolatetissue for transfer to a fresh dish containing 25 ml ice-cold Hanksbuffered saline solution. The tissue section was washed by vigorousshaking and the HBSS replaced. This was repeated two further times oruntil all visible mucus was removed. Mucosa was measured, weighed anddiced into 1 mm2 sections. The tissues sections were transferred to a 50ml polypropylene centrifuge tube containing 50 ml of A52 media(Biosource) supplemented with 2 mM L-glutamine and 1.5 mg/ml dispase(Roche Biochemicals). The digest was incubated for 1 h at 37° C. withfrequent agitation. Following the incubation, the suspension was pouredthrough a 40-mesh cell sieve situated in the base of a 15 cm culturedish. The filtrate was diluted to 50 ml using A52 media supplementedwith 2 mM L-glutamine and passed through a 200-mesh cell sieve. Thefiltrate was collected into a 50 ml polypropylene centrifuge tube andthe suspension was triturated several times followed by vortexing for 2min at setting 6. The density and viability of nucleated cells wasdetermined by flow cytometry using propidium iodide as a negative stainfor viability (Guava system). Erythrocytes were lysed using a standardammonium chloride lysis protocol with incubation at room temperature for10 s. Cells were harvested by centrifugation at 500 g for 5 min at 4° C.The cell pellet was resuspended in 50 ml of ice-cold HBSS andrecentrifuged. The final cell pellet was resuspended in 3 ml of ice-coldHBSS supplemented with 0.1% BSA and 0.25M EDTA. Cell density andviability were estimated using the Guava system and the density adjustedto 1×10⁷ cells per ml. Epithelial cells were stained with a FITC-labeledanti-EpCAM murine monoclonal antibody and enriched by cell sorting usingflow cytometry.

ii) Enrichment of Tumor Cells from Cancer Tissue

Cancer tissue was transferred from the transport vessel to a steriledish containing 25 ml of ice-cold transport buffer. The tissue wasmeasured, weighed and photographed. The tissue was dissected to removenecrotic and fibrotic tissue plaques and the tumour tissue transferredto a fresh dish containing 25 ml ice-cold Hanks buffered salinesolution. The tissue section was washed by vigorous shaking and the HBSSreplaced. This was repeated 2 further times or until all visible mucuswas removed. Tumor tissue was measured, weighed and extensively diced.The tissues slurry was transferred to a 50 ml polypropylene centrifugetube containing 50 ml of A52 media (Biosource) supplemented with 2 mML-glutamine and 1.5 mg/ml dispase (Roche Biochemicals). The digest wasincubated for 1 h at 37° C. with frequent agitation. Following theincubation, the suspension was poured through a 40-mesh cell sievesituated in the base of a 15 cm culture dish. The filtrate was dilutedto 50 ml using A52 media supplemented with 2 mM L-glutamine and passedthrough a 200-mesh cell sieve. The filtrate was collected into a 50 mlpolypropylene centrifuge tube and the suspension was triturated severaltimes followed by vortexing for 2 min at setting 6. The density andviability of nucleated cells was determined by flow cytometry usingpropidium iodide as a negative stain for viability (Guava system).Erythrocytes were lysed using a standard ammonium chloride lysisprotocol with incubation at room temperature for 10 s. Cells wereharvested by centrifugation at 500 g for 5 min at 4° C. The cell pelletwas resuspended in 50 ml of ice-cold HBSS and recentrifuged. The finalcell pellet was resuspended in 3 ml of ice-cold HBSS supplemented with0.1% BSA and 0.25M EDTA. Cell density and viability were estimated usingthe Guava system and the density adjusted to 1×10⁷ cells per ml.Epithelial cells were stained with a FITC-labeled anti-EpCAM murinemonoclonal antibody and enriched by cell sorting using flow cytometry.

iii) Enrichment of Cell Surface Proteins from Sorted Epithelial andTumor Cells

Sorted cells were centrifuged at 500 g at 4° C. for 5 min andresuspended in 50 ml of ice-cold DPBS. The cell suspension was washed by2 further cycles of centrifugation 500 g at 4° C. for 5 min andresuspension of the cell pellet in 50 ml of ice-cold DPBS. Finally, thecell pellet was resuspended in 9.5 ml of ice-cold DPBS and sodiummetaperiodate added to a final concentration of 1 mM. The cellsuspension was incubated on ice for 10 min with frequent agitation inthe dark. Cells were centrifuged at 500 g at 4° C. for 5 min andresuspended in 50 ml of ice-cold DPBS. The cell suspension was washed by2 further cycles of centrifugation 500 g at 4° C. for 5 min andresuspension of the cell pellet in 50 ml of ice-cold DPBS. Finally, thecell pellet was resuspended in lysis buffer (1% SDS [w/v]; 0.1M HEPES;10 mM MgCl₂; 0.1% Non ionic detergent P40; 100 ml protease inhibitorcocktail [P8340, Sigma]) and homogenisation performed by passage oflysate through a 18 G syringe needle 10 times. Protein concentrationswere assayed relative to a Bovine serum albumin standard by a modifiedLowry assay (DC assay, BioRAD) and 1 mg of total cellular proteintransferred to a fresh tube and diluted to 1 mg/ml in acetate buffer(0.1M, pH 5.0).

Cancer Cell Lines:

The model system employed here involves the use of a “normal” reference(i.e., control) to which cell surface expression in tumor-derived celllines is compared. These differentials or candidates are then validatedin normal tissues and cancer tissues to confirm that they aredifferentially expressed between these tissues as well as within thecell line model system.

Cancer Cell Line Culture

Cell lines were grown in a culturing medium that is supplemented asnecessary with growth factors and serum, in accordance with the AmericanType Culture Collection (ATCC) (Manassas, Va.) guidelines for eachparticular cell line. Cultures were established from frozen stocks inwhich the cells were suspended in a freezing medium (cell culture mediumwith 10% DMSO [v/v]) and flash frozen in liquid nitrogen. Frozen stocksprepared in this way were stored in the liquid nitrogen vapour. Cellcultures were established by rapidly thawing frozen stocks at 37° C.Thawed stock cultures were slowly transferred to a culture vesselcontaining a large volume of culture medium that was supplemented. Formaintenance of culture, cells were seeded at 1×10⁵ cells/per ml inmedium and incubated at 37° C. until confluence of cells in the culturevessel exceeds 50% by area. At this time, cells were harvested from theculture vessel using enzymes or EDTA where necessary. The density ofharvested, viable cells was estimated by hemocytometry and the culturereseeded as above. A passage of this nature was repeated no more than 25times at which point the culture was destroyed and reestablished fromfrozen stocks as described above.

For the analyses of cell surface protein expression in cultured celllines, cells were grown as described above. At a period 24 h prior tothe experiment, the cell line was passaged as described above. Thisyielded cell densities that were <50% confluent and growingexponentially. Typically, triplicate analyses of differential expressionwere performed for each line relative to Caco2 for the purpose ofidentifying statistically significant reproducible differentiallyexpressed proteins.

2. Cloning and Expression of Target Proteins

cDNA Retrieval

Peptide sequences were searched by BlastP against the Celera DiscoverySystem (CDS) and public database to identify the correspondingfull-length open reading frames (ORFs). Each ORF sequence was thensearched by BlastN against the Celera in-house human cDNA clonecollection. For each sequence of interest, up to three clones are pulledand streaked onto LB/Ampicillin (100 ug/ml) plates. Plasmid DNA isisolated using Qiagen spin mini-prep kit and verified by restrictiondigest. Subsequently, the isolated plasmid DNA is sequence verifiedagainst the ORF reference sequence. Sequencing reactions are carried outusing Applied Biosystems BigDye Terminator kit followed by ethanolprecipitation. Sequence data is collected using the Applied Biosystems3100 Genetic Analyzer and analyzed by alignment to the referencesequence using the Clone Manager alignment tool.

PCR

PCR primers are designed to amplify the full-length ORF as well as anyregions of the ORF that are interest for expression (antigenic orhydrophilic regions as determined by the Clone Manager sequence analysistool). Primers also contain 5′ and 3′ overhangs to facilitate cloning(see below). PCR reactions contain 2.5 units Platinum Taq DNA PolymeraseHigh Fidelity (Invitrogen), 50 ng cDNA plasmid template, 1 uM forwardand reverse primers, 800 uM dNTP cocktail (Applied Biosystems) and 2 mMMgSO4. After 20-30 cycles (94° C. for 30 seconds, 55° C. for 1 minutesand 73° C. for 2 minutes), product is verified and quantitated byagarose gel electrophoresis.

Construction of Entry Clones

PCR products are cloned into an entry vector for use with the Gatewayrecombination based cloning system (Invitrogen). These vectors includepDonr221, pDonr201, pEntr/D-TOPO or pEntr/SD/D-TOPO and are used asdescribed in the cloning methods below.

TOPO Cloning into pEntr/D-TOPO or pEntr/SD/D-TOPO

For cloning using this method, the forward PCR primer contained a 5′overhang containing the sequence “CACC”. PCR products are generated asdescribed above and cloned into the entry vector using the InvitrogenTOPO cloning kit. Reactions are typically carried out at roomtemperature for 10 minutes and subsequently transformed into TOP10chemically competent cells (Invitrogen, CA). Candidate clones arepicked, plasmid DNA is prepared using Qiagen spin mini-prep kit andscreened using restriction digest. Inserts are subsequently sequenceverified as described above.

Gateway Cloning into pDonr201 or pDonr221

For cloning using this method, PCR primers contained the followingoverhangs:

Forward 5′ overhang: (SEQ ID NO: 186)5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3′ Reverse 5′ overhang:(SEQ ID NO: 187) 5′-GGGGACCACTTTGTACAAGAAAGCTGGGT-3′

PCR products are generated as described above. ORFs are recombined intothe entry vector using the Invitrogen Gateway BP Clonase enzyme mix.Reactions are typically carried out at 25° C. for 1 hour, treated withProteinase K at 37° C. for 10 minutes and transformed into LibraryEfficiency DH5α chemically competent cells (Invitrogen, CA). Candidateclones are picked, plasmid DNA is prepared using Qiagen spin mini-prepkit and screened using restriction digest. Inserts are subsequentlysequence verified as described above.

Construction of Expression Clones

ORFs are transferred from the entry construct into a series ofexpression vectors using the Gateway LR Clonase enzyme mix. Reactionsare typically carried out for 1 hour at 25° C., treated with ProteinaseK at 37° C. for 10 minutes and subsequently transformed into LibraryEfficiency DH5a chemically competent cells (Invitrogen). Candidateclones are picked, plasmid DNA is prepared using Qiagen spin mini-prepkit and screened using restriction digest. Expression vectors includebut are not limited to pDest14, pDest15, pDest17, pDest8, pDest10 andpDest20. These vectors allow expression in systems such as E. coli andrecombinant baculovirus. Other vectors not listed here allow expressionin yeast, mammalian cells, or in vitro.

Expression of Recombinant Proteins in E. coli

Constructs are transformed into one or more of the following hoststrains: BL21 S1, BL21 AI, (Invitrogen); Origami B (DE3), Origami B(DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS, Rosetta-Gami (DE3),Rosetta-Gami (DE3) pLysS, or Rosetta-Gami B (DE3) pLysS (Novagen). Thetransformants are grown in LB with or without NaCl and with appropriateantibiotics, at temperatures in the range of 20-37° C., with aeration.Expression is induced with the addition of IPTG (0.03-0.3 mM) or NaCl(75-300 mM) when the cells are in mid-log growth. Growth is continuedfor one to 24 hours post-induction. Cells are harvested bycentrifugation in a Sorvall RC-3C centrifuge in a H6000A rotor for 10minutes at 3000 rpm, at 4° C. Cell pellets are stored at −80° C.

Expression of Recombinant Proteins Using Baculovirus

Recombinant proteins are expressed using baculovirus in Sf21 fall armyworm ovarian cells. Recombinant baculoviruses are prepared using theBac-to-Bac system (Invitrogen) per the manufacturer's instructions.Proteins are expressed on the large scale in Sf900II serum-free medium(Invitrogen) in a 10 L bioreactor tank (27° C., 130 rpm, 50% dissolvedoxygen for 48 hours).

3. Recombinant Protein Purification

Recombinant proteins are purified from E. coli and/or insect cells usinga variety of standard chromatography methods. Briefly, cells are lysedusing sonication or detergents. The insoluble material is pelleted bycentrifugation at 10,000×g for 15 minutes. The supernatant is applied toan appropriate affinity column, e.g. His-tagged proteins are separatedusing a pre-packed chelating sepharose column (Pharmacia) or GST-taggedproteins are separated using a glutathione sepharose column (Pharmacia).After using the affinity column, proteins are further separated usingvarious techniques, such as ion exchange chromatography (columns fromPharmacia) to separate on the basis of electrical charge or sizeexclusion chromatography (columns from Tosohaas) to separate on thebasis of molecular weight, size and shape.

Expression and purification of the protein are also achieved usingeither a mammalian cell expression system or an insect cell expressionsystem. The pUB6/V5-His vector system (Invitrogen, CA) is used toexpress GSCC in CHO cells. The vector contains the selectable bsd gene,multiple cloning sites, the promoter/enhancer sequence from the humanubiquitin C gene, a C-terminal V5 epitope for antibody detection withanti-V5 antibodies, and a C-terminal polyhistidine (6.times.His)sequence for rapid purification on PROBOND resin (Invitrogen, CA).Transformed cells are selected on media containing blasticidin.

Spodoptera frugiperda (Sf9) insect cells are infected with recombinantAutographica californica nuclear polyhedrosis virus (baculovirus). Thepolyhedrin gene is replaced with the cDNA by homologous recombinationand the polyhedrin promoter drives cDNA transcription. The protein issynthesized as a fusion protein with 6×his which enables purification asdescribed above. Purified protein is used in the following activity andto make antibodies

4. Chemical Synthesis of Peptides

Proteins or portions thereof may be produced not only by recombinantmethods, but also by using chemical methods well known in the art. Solidphase peptide synthesis may be carried out in a batchwise or continuousflow process which sequentially adds α-amino- and side chain-protectedamino acid residues to an insoluble polymeric support via a linkergroup. A linker group such as methylamine-derivatized polyethyleneglycol is attached to poly(styrene-co-divinylbenzene) to form thesupport resin. The amino acid residues are N-a-protected by acid labileBoc (t-butyloxycarbonyl) or base-labile Fmoc(9-fluorenylmethoxycarbonyl). The carboxyl group of the protected aminoacid is coupled to the amine of the linker group to anchor the residueto the solid phase support resin. Trifluoroacetic acid or piperidine areused to remove the protecting group in the case of Boc or Fmoc,respectively. Each additional amino acid is added to the anchoredresidue using a coupling agent or pre-activated amino acid derivative,and the resin is washed. The full length peptide is synthesized bysequential deprotection, coupling of derivitized amino acids, andwashing with dichloromethane and/or N,N-dimethylformamide. The peptideis cleaved between the peptide carboxy terminus and the linker group toyield a peptide acid or amide. (Novabiochem 1997/98 Catalog and PeptideSynthesis Handbook, San Diego Calif. pp. S1-S20). Automated synthesismay also be carried out on machines such as the 431A peptide synthesizer(ABI). A protein or portion thereof may be purified by preparative highperformance liquid chromatography and its composition confirmed by aminoacid analysis or by sequencing (Creighton (1984) Proteins, Structuresand Molecular Properties, W H Freeman, New York N.Y.).

5. Antibody Development

Polyclonal Antibody Preparations:

Polyclonal antibodies against recombinant proteins are raised in rabbits(Green Mountain Antibodies, Burlington, Vt.). Briefly, two New Zealandrabbits are immunized with 0.1 mg of antigen in complete Freund'sadjuvant. Subsequent immunizations are carried out using 0.05 mg ofantigen in incomplete Freund's adjuvant at days 14, 21 and 49. Bleedsare collected and screened for recognition of the antigen by solid phaseELISA and western blot analysis. The IgG fraction is separated bycentrifugation at 20,000×g for 20 minutes followed by a 50% ammoniumsulfate cut. The pelleted protein is resuspended in 5 mM Tris andseparated by ion exchange chromatography. Fractions are pooled based onIgG content. Antigen-specific antibody is affinity purified using PierceAminoLink resin coupled to the appropriate antigen.

Isolation of Antibody Fragments Directed Against CAT from A Library ofscFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againsta CAT to which the donor may or may not have been exposed (see e.g.,U.S. Pat. No. 5,885,793 incorporated herein by reference in itsentirety).

Rescue of the Library: A library of scFvs is constructed from the RNA ofhuman PBLs as described in PCT publication WO 92/01047. To rescue phagedisplaying antibody fragments, approximately 10⁹ E. coli harboring thephagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and100.mu.g/ml of ampicillin (2.times.TY-AMP-GLU) and grown to an O.D. of0.8 with shaking. Five ml of this culture is used to innoculate 50 ml of2.times.TY-AMP-GLU, 2×10⁸ TU of delta gene 3 helper (M13 delta gene III,see PCT publication WO 92/01047) are added and the culture incubated at37° C. for 45 minutes without shaking and then at 37° C. for 45 minuteswith shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. andthe pellet resuspended in 2 liters of 2×TY containing 100.mu.g/mlampicillin and 50 ug/ml kanamycin and grown overnight. Phage areprepared as described in PCT publication WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2×TY broth containing 100.mu.g ampicillin/ml and25.mu.g kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37°C. Phagre particles are purified and concentrated from the culturemedium by two PEG-precipitations (Sambrook et al., 2001), resuspended in2 ml PBS and passed through a 0.45.mu.m filter (Minisart NML; Sartorius)to give a final concentration of approximately 1013 transducing units/ml(ampicillin-resistant clones).

Panning of the Library: Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100.mu.g/ml or 10.mu.g/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phages are then used to infect 10 ml of mid-log E.coli TG1 by incubating eluted phage with bacteria for 30 minutes at 37°C. The E. coli are then plated on TYE plates containing 1% glucose and100.mu.g/ml ampicillin. The resulting bacterial library is then rescuedwith delta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders: Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10.mu.g/ml ofthe polypeptide of the present invention in 50 mM bicarbonate pH 9.6.Clones positive in ELISA are further characterized by PCR fingerprinting(see, e.g., PCT publication WO 92/01047) and then by sequencing.

Monoclonal Antibody Generation

i) Materials:

1) Complete Media No Sera (CMNS) for washing of the myeloma and spleencells; Hybridoma medium CM-HAT {Cell Mab (BD), 10% FBS (or HS); 5%Origen HCF (hybridoma cloning factor) containing 4 mM L-glutamine andantibiotics} to be used for plating hybridomas after the fusion.

2) Hybridoma medium CM-HT (NO AMINOPTERIN) (Cell Mab (BD), 10% FBS 5%Origen HCF containing 4 mM L-glutamine and antibiotics) to be used forfusion maintenance are stored in the refrigerator at 4-6° C. The fusionsare fed on days 4, 8, and 12, and subsequent passages. Inactivated andpre-filtered commercial Fetal Bovine serum (FBS) or Horse Serum (HS) arethawed and stored in the refrigerator at 4° C. and must be pretested formyeloma growth from single cells.

3) The L-glntamine (200 mM, 100× solution), which is stored at −20° C.freezer, is thawed and warmed until completely in solution. TheL-glntamin is dispensed into media to supplement growth. L-glntamin isadded to 2 mM for myelomas, and 4 mM for hybridoma media. Further thePenicillin, Streptomycin, Amphotericin (antibacterial-antifungal storedat −20° C.) is thawed and added to Cell Mab Media to 1%.

4) Myeloma growth media is Cell Mab Media (Cell Mab Media, Quantum Yieldfrom BD is stored in the refrigerator at 4° C. in the dark) which areadded L-glntamine to 2 mM and antibiotic/antimycotic solution to 1% andis called CMNS.

5) 1 bottle of PEG 1500 in Hepes (Roche, N.J.)

6) 8-Azaguanine is stored as the dried powder supplied by SIGMA at −700°C. until needed. Reconstitute 1 vial/500 ml of media and add entirecontents to 500 ml media (eg. 2 vials/liter).

7) Myeloma Media is CM which has 10% FBS (or HS) and 8-Aza (1×) storedin the refrigerator at 4° C.

8) Clonal cell medium D (Stemcell, Vancouver) contains HAT and methylcellulose for semi-solid direct cloning from the fusion. This comes in90 ml bottles with a CoA and must be “melted at 37° C. in a waterbath inthe morning of the day of the fusion. Loosen the cap and leave in CO₂incubator to sufficiently gas the medium D and bring the pH down.

9) Hybridoma supplements HT [hypoxanthine, thymidine] are to be used inmedium for the section of hybridomas and maintenace of hybridomasthrough the cloning stages respectively.

10) Origen HCF can be obtained directly from Igen and is a cellsupernatant produced from a macrophage-like cell-line. It can be thawedand aliqouted to 15 ml tubes at 5 ml per tube and stored frozen at −20°C. Positive Hybridomas are fed HCF through the first subcloning and aregradually weaned. It is not necessary to continue to supplement unlessyou have a particularly difficult hybridoma clone. This and otheradditives have been shown to be more effective in promoting newhybridoma growth than conventional feeder layers.

ii) Procedure

To generate monoclonal antibodies, mice are immunized with 5-50 ug ofantigen either intra-peritoneally (i.p.) or by intravenous injection inthe tail vein (i.v.). Typically, the antigen used is a recombinantprotein that is generated as described above. The primary immunizationtakes place 2 months prior to the harvesting of splenocytes from themouse and the immunization is typically boosted by i.v. injection of5-50 ug of antigen every two weeks. At least one week prior to expectedfusion date, a fresh vial of myeloma cells is thawed and cultured.Several flasks at different densities are maintained in order that aculture at the optimum density is ensured at the time of fusion. Theoptimum density is determined to be 3−6×10⁵ cells/ml. Two to five daysbefore the scheduled fusion, a final immunization is administered of ˜5ug of antigen in PBS i.p. or i.v.

Myeloma cells are washed with 30 ml serum free media by centrifugationat 500 g at 4° C. for 5 minutes. Viable cell density is determined inresuspended cells using hemocytometry and vital stains. Cellsresuspended in complete growth medium are stored at 37° C. during thepreparation of splenocytes. Meanwhile, to test aminopterin sensitivity,1×10⁶ myeloma cells are transferred to a 15 ml conical tube andcentrifuged at 500 g at 4° C. for 5 minutes. The resulting pellet isresuspended in 15 ml of HAT media and cells plated at 2 drops/well on a96 well plate.

To prepare splenocytes from immunized mice, the animals are euthanisedand submerged in 70% ETOH. Under sterile conditions, the spleen issurgically removed and placed in 10 ml of RPMI medium supplemented with20% fetal calf serum in a Petri dish. Cells are extricated from thespleen by infusing the organ with medium >50 times using a 21 g syringe.

Cells are harvested and washed by centrifugation (at 500 g at 4° C. for5 minutes) with 30 ml of medium. Cells are resuspended in 10 ml ofmedium and the density of viable cells determined by hemocytometry usingvital stains. The splenocytes are mixed with myeloma cells at a ratio of5:1 (spleen cells: myeloma cells). Both the myeloma and spleen cells arewashed 2 more times with 30 ml of RPMI-CMNS. Spin at 800 rpm for 12minutes.

Supernatant is removed and cells are resuspended in 5 ml of RPMI-CMNSand are pooled to fill volume to 30 ml and spin down as before. Then,the pellet is broken up by gently tapping on the flow hood surface andresuspended in 1 ml of BMB REG1500 (prewarmed to 37° C.) dropwise with 1cc needle over 1 minute.

RPMI-CMNS to the PEG cells and RPMI-CMNS are added to slowly dilute outthe PEG. Cells are centrifuged and diluted in 5 ml of Complete media and95 ml of Clonacell Medium D (HAT) media (with 5 ml of HCF). The cellsare plated out 10 ml per small petri plate.

Myeloma/HAT control. P is prepared as follows: dilute about 1000 P3X63Ag8.653 myeloma cells into 1 ml of mediu D and transfer into a singlewell of a 24 well plate. Plates are placed in incubator, with two platesinside of a large petri plate, with an additional petri plate full ofdistilled water, for 10-18 days under 5% CO2 overlay at 37° C. Clonesare picked from semisolid agarose into 96 well plates containing 150-200ul of CM-HT. Supernatants are screened 4 days later in ELISA, andpositive clones are moved up to 24 well plates. Heavy growth willrequire changing of the media at day 8 (+/−150 ml). One should furtherdecrease the HCF to 0.5% (gradually-2%, then 1%, then 0.5%) in thecloning plates.

For further references see Kohler G, and C. Milstein Continuous culturesof fused cells secreting antibody of predefined specificity. 1975.Nature 256: 495-497; Lane, R. D. A short duration polyethylene glycolfusion technique for increasing production of monoclonalantibody-secreting hybridomas. 1985. J. Immunol. Meth. 81:223-228;

Harlow, E. and D. Lane. Antibodies: A laboratory manual. Cold SpringHarbour Laboratory Press. 1988; Kubitz, D. The Scripps ResearchInstitute. La Jolla. Personal Communication; Zhong, G., Berry, J. D.,and Choukri, S. (1996) Mapping epitopes of Chlamydia trachomatisneutralizing monoclonal antibodies using phage random peptide libraries.J. Indust. Microbiol. Biotech. 19, 71-76; Berry, J. D., Licea, A.,Popkov, M., Cortez, X., Fuller, R., Elia, M., Kerwin, L., and C. F.Barbas III. (2003) Rapid monoclonal antibody generation via dendriticcell targeting in vivo. Hybridoma and Hybridomics 22 (1), 23-31.

6. mRNA Expression

Validation in Tissues by Taqman

Expression of mRNA is quantitated by RT-PCR using TaqMan® technology.The Taqman system couples a 5′ fluorogenic nuclease assay with PCR forreal time quantitation. A probe is used to monitor the formation of theamplification product.

Total RNA is isolated from disease model cell lines using the RNEasyKit® (Qiagen) per manufacturer's instructions and included DNasetreatment. Normal human tissue RNAs are acquired from commercial vendors(Ambion, Austin, Tex.; Stratagene, La Jolla, Calif., BioChain Institute,Newington, N.H.) as were RNAs from matched disease/normal tissues.

Target transcript sequences are identified for the differentiallyexpressed peptides by searching the BlastP database. TaqMan assays (PCRprimer/probe set) specific for those transcripts are identified bysearching the Celera Discovery System™ (CDS) database. The assays aredesigned to span exon-exon borders and do not amplify genomic DNA.

The TaqMan primers and probe sequences are as designed by AppliedBiosystems (AB) as part of the Assays on Demand™ product line or bycustom design through the AB Assays by Design^(SM) service.

RT-PCR is accomplished using AmpliTaqGold and MultiScribe reversetranscriptase in the One Step RT-PCR Master Mix reagent kit (AB)according to the manufacturer's instructions. Probe and primerconcentrations are 250 nM and 900 nM, respectively, in a 15 μl reaction.For each experiment, a master mix of the above components is made andaliquoted into each optical reaction well. Eight nanograms of total RNAis the template. Each sample is assayed in triplicate. QuantitativeRT-PCR is performed using the ABI Prism® 7900HT Sequence DetectionSystem (SDS). Cycling parameters follow: 48° C. for 30 min. for onecycle; 95° C. for 10 min for one cycle; 95° C. for 15 sec, 60° C. for 1min. for 40 cycles.

The SDS software calculates the threshold cycle (C_(T)) for eachreaction, and C_(T) values are used to quantitate the relative amount ofstarting template in the reaction. The C_(T) values for each set ofthree reactions are averaged for all subsequent calculations

Data are analyzed for fold difference in expression using an endogenouscontrol for normalization and is expressed relative to a normal tissueor normal cell line reference. The choice of endogenous control isdetermined empirically by testing various candidates against the cellline and tissue RNA panels and selecting the one with the leastvariation in expression. Relative changes in expression are quantitatedusing the 2^(−ΔΔCT) Method. Livak, K. J. and Schmittgen, T. D. (2001)Methods 25: 402-408; User bulletin #2: ABI Prism 7700 Sequence DetectionSystem.

Validation by Tissue Flow Cytometry Analysis

Post tissue processing, cells are sorted by flow cytometry known in theart to enrich for epithelial cells. Alternatively, cells isolated fromlung tissue are stained directly with EpCAM (for epithelial cells) andthe specific antibody to a CAT. Cell numbers and viability aredetermined by PI exclusion (GUAVA) for cells isolated from both normaland tumor tissue. A minimum of 0.5×10⁶ cells are used for each analysis.Cells are washed once with Flow Staining Buffer (0.5% BSA, 0.05% NaN₃ inD-PBS). To the cells, 20 ul of each antibody for CAT are added. Anadditional 5 ul of EpCAM antibody conjugated to APC were added whenunsorted cells are used in the experiment. Cells are incubated withantibodies for 30 minutes at 4° C. Cells are washed once with FlowStaining Buffer and either analyzed immediately on the LSR flowcytometry apparatus or fixed in 1% formaldehyde and store at 4° C. untilLSR analysis. Antibodies used to detect a CAT may be purchased from BDBiosciences and PE-conjugated. The isotype control antibody used forthese experiments is PE-conjugated mouse IgG1k.

7. Detection and Diagnosis of CAT by Liquid Chromatography and MassSpectrometry (LC/MS)

The proteins from cells can be prepared by methods known in the art (R.Aebersold Nature Biotechnology Volume 21 Number 6 Jun. 2003).

The differential expression of proteins in disease and healthy samplesare quantitated using Mass Spectrometry and ICAT (Isotope Coded AffinityTag) labeling, which is known in the art. ICAT is an isotope labeltechnique that allows for discrimination between two populations ofproteins, such as a healthy and a disease sample that are pooledtogether for experimental purposes or two acquisitions of the samesample for classification of true sample peptides from LC/MS noiseartifacts. The LC/MS spectra are collected for the labeled samples andprocessed using the following steps:

The raw scans from the LC/MS instrument are subjected to peak detectionand noise reduction software. Filtered peak lists are then used todetect ‘features’ corresponding to specific peptides from the originalsample(s). Features are characterized by their mass/charge, charge,retention time, isotope pattern and intensity.

Similar experiments are repeated in order to increase the confidence indetection of a peptide. These multiple acquisitions are computationallyaggregated into one experiment. Experiments involving healthy anddisease samples used the known effects of the ICAT label to classify thepeptides as originating from a particular sample or from both samples.The intensity of a peptide present in both healthy and disease samplesis used to calculate the differential expression, or relative abundance,of the peptide. The intensity of a peptide found exclusively in onesample is used to calculate a theoretical expression ratio for thatpeptide (singleton). Expression ratios are calculated for each peptideof each replicate of the experiment.

Statistical tests are performed to assess the robustness of the data andselect statistically significant differentials. To assess generalquality of the data, one: a) ensured that similar features are detectedin all replicates of the experiment; b) assessed the distribution of thelog ratios of all peptides (a Gaussian is expected); c) calculated theoverall pair wise correlations between ICAT LC/MS maps to ensure thatthe expression ratios for peptides are reproducible across the multiplereplicates; d) aggregated multiple experiments in order to compare theexpression ratio of a peptide in multiple diseases or disease samples.

8. Expression Validation by Immunohistochemistry (IHC) in TissueSections

Tissue Sections

Paraffin embedded, fixed tissue sections are obtained from a panel ofnormal tissues (Adrenal, Bladder, Lymphocytes, Bone Marrow, Breast,Cerebellum, Cerebral cortex, Colon, Endothelium, Eye, Fallopian tube,Small Intestine, Heart, Kidney [glomerulus, tubule], Liver, Lung, Testesand Thyroid) as well as 30 tumor samples with matched normal adjacenttissues from pancreas, lung, colon, prostate, ovarian and breast. Inaddition, other tissues are selected for testing such as bladder renal,hepatocellular, pharyngeal and gastric tumor tissues. Replicate sectionsare also obtained from numerous tumor types (Bladder Cancer, LungCancer, Breast Cancer, Melanoma, Colon Cancer, Non-Hodgkins Lymphoma,Endometrial Cancer, Ovarian Cancer, Head and Neck Cancer, ProstateCancer, Leukemia [ALL and CML] and Rectal Cancer). Sections are stainedwith hemotoxylin and eosin and histologically examined to ensureadequate representation of cell types in each tissue section.

An identical set of tissues will be obtained from frozen sections andare used in those instances where it is not possible to generateantibodies that are suitable for fixed sections. Frozen tissues do notrequire an antigen retrieval step.

Paraffin Fixed Tissue Sections

Hemotoxylin and Eosin staining of paraffin embedded, fixed tissuesections.

Sections are deparaffinized in 3 changes of xylene or xylene substitutefor 2-5 minutes each. Sections are rinsed in 2 changes of absolutealcohol for 1-2 minutes each, in 95% alcohol for 1 minute, followed by80% alcohol for 1 minute. Slides are washed well in running water andstained in Gill solution 3 hemotoxylin for 3 to 5 minutes. Following avigorous wash in running water for 1 minute, sections are stained inScott's solution for 2 minutes. Sections are washed for 1 min in runningwater then conterstained in Eosin solution for 2-3 minutes dependingupon development of desired staining intensity. Following a brief washin 95% alcohol, sections are dehydrated in three changes of absolutealcohol for 1 minute each and three changes of xylene or xylenesubstitute for 1-2 minutes each. Slides are coverslipped and stored foranalysis.

Optimisation of Antibody Staining

For each antibody, a positive and negative control sample are generatedusing data from the ICAT analysis of the cancer cell lines/tissues.Cells are selected that are known to express low levels of a particulartarget as determined from the ICAT data. This cell line is the referencenormal control. Similarly, a cancer cell line that is determined toover-express the target is selected.

Antigen Retrieval

Sections are deparaffinized and rehydrated by washing 3 times for 5minutes in xylene; two times for 5 minutes in 100% ethanol; two timesfor 5 minutes in 95% ethanol; and once for 5 minutes in 80% ethanol.Sections are then placed in endogenous blocking solution (methanol+2%hydrogen peroxide) and incubated for 20 minutes at room temperature.Sections are rinsed twice for 5 minutes each in deionized water andtwice for 5 minutes in phosphate buffered saline (PBS), pH 7.4.Alternatively, where necessary sections are deparrafinized by HighEnergy Antigen Retrieval as follows: sections are washed three times for5 minutes in xylene; two times for 5 minutes in 100% ethanol; two timesfor 5 minutes in 95% ethanol; and once for 5 minutes in 80% ethanol.Sections are placed in a Coplin jar with dilute antigen retrievalsolution (10 mM citrate acid, pH 6). The Coplin jar containing slides isplaced in a vessel filled with water and microwaved on high for 2-3minutes (700 watt oven). Following cooling for 2-3 minutes, steps 3 and4 are repeated four times (depending on tissue), followed by cooling for20 minutes at room temperature. Sections are then rinsed in deionizedwater, two times for 5 minutes, placed in modified endogenous oxidationblocking solution (PBS+2% hydrogen peroxide). and rinsed for 5 minutesin PBS.

Blocking and Staining

Sections are blocked with PBS/1% bovine serum albumin (PBA) for 1 hourat room temperature followed by incubation in normal serum diluted inPBA (2%) for 30 minutes at room temperature to reduce non-specificbinding of antibody. Incubations are performed in a sealed humiditychamber to prevent air-drying of the tissue sections. (The choice ofblocking serum is the same as the species of the biotinylated secondaryantibody). Excess antibody is gently removed by shaking and sectionscovered with primary antibody diluted in PBA and incubated either atroom temperature for 1 hour or overnight at 4° C. (Care is taken thatthe sections do not touch during incubation). Sections are rinsed twicefor 5 minutes in PBS, shaking gently. Excess PBS is removed by gentlyshaking. The sections are covered with diluted biotinylated secondaryantibody in PBA and incubated for 30 minutes to 1 hour at roomtemperature in the humidity chamber. If using a monoclonal primaryantibody, addition of 2% rat serum is used to decrease the background onrat tissue sections. Following incubation, sections are rinsed twice for5 minutes in PBS, shaking gently. Excess PBS is removed and sectionsincubated for 1 hour at room temperature in Vectastain ABC reagent (asper kit instructions). The lid of the humidity chamber is secured duringall incunations to ensure a moist environment. Sections are rinsed twicefor 5 minutes in PBS, shaking gently.

Develop and Counterstain

Sections are incubated for 2 minutes in peroxidase substrate solutionthat is made up immediately prior to use as follows:

-   -   10 mg diaminobenzidine (DAB) dissolved in 10 ml 50 mM sodium        phosphate buffer, pH 7.4.    -   12.5 microliters 3% CoCl2/NiCl2 in deionized water    -   1.25 microliters hydrogen peroxide

Slides are rinsed well three times for 10 min in deionized water andcounterstained with 0.01% Light Green acidified with 0.01% acetic acidfor 1-2 minutes depending on intensity of counterstain desired.

Slides are rinsed three times for 5 minutes with deionized water anddehydrated two times for 2 minutes in 95% ethanol; two times for 2minutes in 100% ethanol; and two times for 2 minutes in xylene. Stainedslides are mounted for visualization by microscopy.

9. IHC Staining of Frozen Tissue Sections

Fresh tissues are embedded carefully in OCT in plastic mold, withouttrapping air bubbles surrounding the tissue. Tissues are frozen bysetting the mold on top of liquid nitrogen until 70-80% of the blockturns white at which point the mold is placed on dry ice. The frozenblocks were stored at −80° C. Blocks are sectioned with a cryostat withcare taken to avoid warming to greater than −10° C. Initially, the blockis equilibrated in the cryostat for about 5 minutes and 6-10 mm sectionsare cut sequentially. Sections are allowed to dry for at least 30minutes at room temperature. Following drying, tissues are stored at 4°C. for short term and −80° C. for long term storage.)

Sections are fixed by immersing in acetone jar for 1-2 minutes at roomtemperature, followed by drying at room temp. Primary antibody is added(diluted in 0.05 M Tris-saline [0.05 M Tris, 0.15 M NaCl, pH 7.4], 2.5%serum) directly to the sections by covering the section dropwise tocover the tissue entirely. Binding is carried out by incubation achamber for 1 hour at room temperature. Without letting the sections dryout, the secondary antibody (diluted in Tris-saline/2.5% serum) is addedin a similar manner to the primary and incubated as before (at least 45minutes).

Following incubation, the sections are washed gently in Tris-saline for3-5 minutes and then in Tris-saline/2.5% serum for another 3-5 minutes.If a biotinylated primary antibody is used, in place of the secondaryantibody incubation, slides are covered with 100 ul of diluted alkalinephosphatase conjugated streptavidin, incubated for 30 minutes at roomtemperature and washed as above. Sections are incubated with alkalinephosphatse substrate (1 mg/ml Fast Violet; 0.2 mg/ml Napthol AS-MXphosphate in Tris-Saline pH 8.5) for 10-20 minutes until the desiredpositive staining is achieved at which point the reaction is stopped bywashing twice with Tris-saline. Slides are counter-stained with Mayer'shematoxylin for 30 seconds and washed with tap water for 2-5 minutes.Sections are mounted with Mount coverslips and mounting media.

10. Assay for Antibody Dependent Cellular Cytotoxicity

Cultured tumor cells are labeled with 100 μCi 51Cr for 1 hour;Livingston, P. O., Zhang, S., Adluri, S., Yao, T.-J., Graeber, L.,Ragupathi, G., Helling, F., & Fleischer, M. (1997). Cancer Immunol.Immunother. 43, 324-330. After being washed three times with culturemedium, cells are resuspended at 10⁵/ml, and 100 μl/well are plated onto96-well round-bottom plates. A range of antibody concentrations areapplied to the wells, including an isotype control together with donorperipheral blood mononuclear cells that are plated at a 100:1 and 50:1ratio. After an 18-h incubation at 37° C., supernatant (30 μl/well) isharvested and transferred onto Lumaplate 96 (Packard), dried, and readin a Packard Top-Count NXT y counter. Each measurement is carried out intriplicate. Spontaneous release is determined by cpm of tumor cellsincubated with medium and maximum release by cpm of tumor cells plus 1%Triton X-100 (Sigma). Specific lysis is defined as: % specificlysis=[(experimental release-spontaneous release)/(maximumrelease-spontaneous release)]×100. The percent ADCC is expressed as peakspecific lysis postimmune subtracted by preimmune percent specificlysis. A doubling of the ADCC to >20% is considered significant.

11. Assay for Complement Dependent Cytotoxicity

Chromium release assays to assess complement-mediated cytotoxicity areperformed for each patient at various time points; Dickler, M. N.,Ragupathi, G., Liu, N. X., Musselli, C., Martino, D. J., Miller, V. A.,Kris, M. G., Brezicka, F. T., Livingston, P. O. & Grant, S. C. (1999)Clin. Cancer Res. 5, 2773-2779. Cultured tumor cells are washed inFCS-free media two times, resuspended in 500 μl of media, and incubatedwith 100 μCi ⁵¹Cr per 10 million cells for 2 h at 37° C. The cells arethen shaken every 15 min for 2 h, washed 3 times in media to achieve aconcentration of approximately 20,000 cells/well, and then plated inround-bottom plates. The plates contain either 50 μl cells plus 50 μlmonoclonal antibody, 500 cells plus serum (pre- and posttherapy), or 50μl cells plus mouse serum as a control. The plates are incubated in acold room on a shaker for 45 min. Human complement of a 1:5 dilution(resuspended in 1 ml of ice-cold water and diluted with 3% human serumalbumin) is added to each well at a volume of 100 μl. Control wellsinclude those for maximum release of isotope in 10% Triton X-100 (Sigma)and for spontaneous release in the absence of complement with mediumalone. The plates are incubated for 2 h at 37° C., centrifuged for 3min, and then 100 μl of supernatant is removed for radioactivitycounting. The percentage of specific lysis is calculated as follows: %cytotoxicity=[(experimental release-spontaneous release)/(maximumrelease-spontaneous release)]×100.A doubling of the CDC to >20% isconsidered significant.

12. In Vitro Assays in Cell Lines

RNAi

Lipofectamine 2000 and Plus were purchased from Invitrogen (Carlsbad,Calif.) and GeneSilencer from Gene Therapy Systems (San Diego, Calif.).Synthetic siRNA oligonucleotides were from Dharmacon (Lafayette, Colo.),Qiagen (Valencia, Calif.). RNeasy 96 Kit was purchased from Qiagen(Valencia, Calif.). Apop-one homogeneous caspase-3/7 kit and CellTiter96 AQueous One solution cell proliferation assay were both purchasedfrom Promega (Madison, Wis.). Alamar Blue proliferation assay waspurchased from Biosource (Camarillo, Calif.).

RNAi Transfections

In the initial screening phase, RNAi was performed by using 100 nM(final) of Smartpools (Dharmacon), pool of 4—for Silencing siRNAduplexes (Qiagen) or non-targeting negative control siRNA (Dharmacon orQiagen). In the breakout phase, each individual duplex was used at 100nM (final). In the titration phase, individual duplex were used at0.1-100 nM (final). Transient transfections were carried out by usingeither Lipofectamine 2000 from Invitrogen (Carlsbad, Calif.) or by usingGeneSilencer from Gene Therapy Systems (San Diego, Calif.) in methodsdescribed below. 1 day after transfections, total RNA was isolated byusing the RNeasy 96 Kit (Qiagen) according to manufacturer'sinstructions and expression of mRNA was quantitated by using TaqMantechnology. Apoptosis and proliferation assays were performed dailyusing Apop-one homogeneous caspase-3/7 kit and Alamar Blue or CellTiter96 AQueous One Solution Cell Proliferation Assays (see below).

RNAi Transfections-Lipofectamine 2000

Transient transfections were carried out on sub-confluent cancer celllines as previously described (Elbashir, S. M. et al. (2001) Nature 411:494-498, Caplen, N. J. et al. (2001) Proc Natl Acad Sci USA 98:9742-9747, Sharp, P. A. (2001) Genes and Development 15: 485-490).Synthetic RNA to gene of interest or non-targeting negative controlsiRNA were transfected using lipofectamine according to manufacturer'sinstructions. Cells were plated in 96 well plates in antibiotics freemedium. The next day, the transfection reagent and siRNA were preparedfor transfections as follows: For each well, 0.1-100 nM siRNA wasresuspended in 25 ul serum-free media with Plus and incubated at roomtemperature for 15 minutes. 0.1-1 ul of lipofectamine 2000 was thenresuspended in serum-free medium. After incubation, the diluted siRNAand the lipofectamine 2000 were combined and incubated for 15 minutes atroom temperature. Media was then removed from the cells and the combinedsiRNA-Lipofectamine 2000 reagent added to a final volume of 50 ul perwell. After a further 4 hours incubation, 50 ul serum containing mediumwas added to each well. 1 and 4 days after transfection, expression ofmRNA was quantitated by RT-PCR using TaqMan technology and proteinexpression levels were examined by flow cytometry. Apoptosis andproliferation assays were performed daily using Apop-one homogeneouscaspase-3/7 kit and Alamar Blue or CellTiter 96 AQueous One SolutionCell Proliferation Assays (see below).

RNAi Transfections-GeneSilencer

Transient transfections were carried out on sub-confluent cancer celllines as previously described. Synthetic RNA to gene of interest orscrambled negative control siRNA were transfected using GeneSilenceraccording to manufacturer's instructions. Cells were plated in 96 wellplates in antibiotics free medium. The next day, the transfectionreagent and the synthetic siRNA were prepared for transfections asfollows: 1-1.5 ul of Gene Silencer was diluted in serum-free media to afinal volume of 20 ul per well. After resuspending 0.1-100 nM siRNA in20 ul serum-free media, the reagents were combined and incubated at roomtemperature for 5-20 minutes. After incubation, the siRNA-Gene Silencerreagent was added to each well to a final volume of 50 ul per well.After further incubation in a 37° C. incubator for 4 hours, an equalvolume of serum containing media was added back to the cultured cells.The cells were then incubated for 1 to 4 days before mRNA, proteinexpression and effects on apoptosis and proliferation were examined.

Apoptosis

Apoptosis assay was performed by using the Apop-one homogeneouscaspase-3/7 kit from Promega. Briefly, the caspase-3/7 substrate wasthawed to room temperature and diluted 1:100 with buffer. The dilutedsubstrate was then added 1:1 to cells, control or blank. The plates werethen placed on a plate shaker for 30 minutes to 18 hours at 300-500 rpm.The fluorescence of each well was then measured at using an excitationwavelength of 485+/−20 nm and an emission wavelength of 530+/−25 nm.

Proliferation—MTS

Proliferation assay was performed by using the CellTiter 96 AQueous OneSolution Cell Proliferation Assay kit from Promega. 20 ul of CellTiter96 AQueous One Solution was added to 100 ul of culture medium. Theplates were then incubated for 1-4 hours at 37° C. in a humidified 5%CO₂ incubator. After incubation, the change in absorbance was read at490 nm.

Proliferation—Alamar Blue

Proliferation assay was performed by using the Alamar Blue assay fromBiosource. 10 ul of Alamar Blue reagent was added to 100 ul of cells inculture medium. The plates were then incubated for 1-4 hours at 37° C.in a humidified 5% CO₂ incubator. After incubation, the change influorescence was measured at using an excitation wavelength of 530 nmand an emission wavelength of 595 nm.

mRNA Expression

Expression of mRNA was quantitated by RT-PCR using TaqMan® technology.Total RNA was isolated from cancer model cell lines using the RNEasy 96kit (Qiagen) per manufacturer's instructions and included DNasetreatment. Target transcript sequences were identified for thedifferentially expressed peptides by searching the BlastP database.TaqMan assays (PCR primer/probe set) specific for those transcripts wereidentified by searching the Celera Discovery System™ (CDS) database. Theassays are designed to span exon-exon borders and do not amplify genomicDNA. The TaqMan primers and probe sequences were as designed by AppliedBiosystems (AB) as part of the Assays on Demand™ product line or bycustom design through the AB Assays by Design^(SM) service. RT-PCR wasaccomplished using AmpliTaqGold and MultiScribe reverse transcriptase inthe One Step RT-PCR Master Mix reagent kit (AB) according to themanufacturers instructions. Probe and primer concentrations were 900 nMand 250 nM, respectively, in a 250 reaction. For each experiment, amaster mix of the above components was made and aliquoted into eachoptical reaction well. 5 ul of total RNA was the template. Each samplewas assayed in triplicate. Quantitative RT-PCR was performed using theABI Prism® 7900HT Sequence Detection System (SDS). Cycling parametersfollow: 48° for 30 min. for one cycle; 95° C. for 10 min for one cycle;95° C. for 15 sec, 60° C. for 1 min. for 40 cycles.

The SDS software calculates the threshold cycle (CT) for each reaction,and CT values were used to quantitate the relative amount of startingtemplate in the reaction. The CT values for each set of three reactionswere averaged for all subsequent calculations.

Total RNA was quantitated by using RiboGreen RNA Quantitation Kitaccording to manufacturer's instructions and the % mRNA expression wascalculated using total RNA for normalization. % knockdown was thencalculated relative to the no addition control.

Testing of Functional Blocking Antibodies

Sub-confluent lung cancer cell lines are serum-starved overnight. Thenext day, serum-containing media is added back to the cells in thepresence of 5-50 ng/ml of function blocking antibodies. After 2 or 5days incubation at 37° C. 5% CO₂, antibody binding is examined by flowcytometry and apoptosis and proliferation are examined by usingprotocols described below.

Cell Invasion

Cell invasion assay is performed by using the 96 well cell invasionassay kit available from Chemicon. After the cell invasion chamberplates are adjusted to room temperature, 100 ul serum-free media isadded to the interior of the inserts. 1-2 hours later, cell suspensionsof 1×10⁶ cells/ml are prepared. Media is then carefully removed from theinserts and 100 ul of prepared cells are added into the insert+/−0 to 50ng function blocking antibodies. The cells are pre-incubated for 15minutes at 37° C. before 150 ul of media containing 10% FBS is added tothe lower chamber. The cells are then incubated for 48 hours at 37° C.After incubation, the cells from the top side of the insert arediscarded and the invasion chamber plates are then placed on a new96-well feeder tray containing 150 ul of pre-warmed cell detachmentsolution in the wells. The plates are incubated for 30 minutes at 37° C.and are periodically shaken. Lysis buffer/dye solution (4 ul CyQuantDye/300 ul 4× lysis buffer) is prepared and added to each well ofdissociation buffer/cells on feeder tray. The plates are incubated for15 minutes at room temperature before 150 ul is transferred to a new96-well plate. Fluorescence of invading cells is then read at 480 nmexcitation and 520 nm emission.

Receptor Internalization

For quantification of receptor internalization, ELISA assays areperformed essentially as described by Daunt et al. (Daunt, D. A., Hurtz,C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997) Mol.Pharmacol. 51, 711-720.) The cell lines are plated at 6×10⁵ cells per ina 24-well tissue culture dishes that have previously been coated with0.1 mg/ml poly-L-lysine. The next day, the cells are washed once withPBS and incubated in DMEM at 37° C. for several minutes. Agonist to thecell surface target of interest is then added at a pre-determinedconcentration in prewarmed DMEM to the wells. The cells are thenincubated for various times at 37° C. and reactions are stopped byremoving the media and fixing the cells in 3.7% formaldehyde/TBS for 5min at room temperature. The cells are then washed three times with TBSand nonspecific binding blocked with TBS containing 1% BSA for 45 min atroom temperature. The first antibody is added at a pre-determineddilution in TBS/BSA for 1 hr at room temperature. Three washes with TBSfollowed, and cells are briefly reblocked for 15 min at roomtemperature. Incubation with goat anti-mouse conjugated alkalinephosphatase (Bio-Rad) diluted 1:1000 in TBS/BSA is carried out for 1 hat room temperature. The cells are washed three times with TBS and acolorimetric alkaline phosphatase substrate is added. When the adequatecolor change is reached, 100-μl samples are taken for colorimetricreadings.

13. In Vivo Studies by Using Antibodies

Treatment of Cancer Cells with Monoclonal Antibodies.

Cancer cells are seeded at a density of 4×10⁴ cells per well in 96-wellmicrotiter plates and allowed to adhere for 2 hours. The cells are thentreated with different concentrations of anti-CAT monoclonal antibody(Mab) or irrelevant isotype matched (anti-rHuIFN-. gamma. Mab) at 0.05,0.5 or 5.0 mug/ml. After a 72 hour incubation, the cell monolayers arestained with crystal violet dye for determination of relative percentviability (RPV) compared to control (untreated) cells. Each treatmentgroup consists of replicates. Cell growth inhibition is monitored.

Treatment of NIH 3T3 Cells Overexpression CAT Protein with MonoclonalAntibodies.

NIH 3T3 expressing a CAT protein are treated with differentconcentrations of anti-CAT MAbs. Cell growth inhibition is monitored.

In Vivo Treatment of NIH 3T3 Cells Overexpressing CAT with Anti-CATMonoclonal Antibodies.

NIH 3T3 cells transfected with either a CAT expression plasmid or theneo-DHFR vector are injected into nu/nu (athymic) mice subcutaneously ata dose of 10⁶ cells in 0.1 ml of phosphate-buffered saline. On days 0,1, 5 and every 4 days thereafter, 100 mug (0.1 ml in PBS) of either anirrelevant or anti-CAT monoclonal antibody of the IG2A subclass isinjected intraperitoneally. Tumor occurrence and size are monitored for1 month period of treatment.

14. Summary of Experimental Validation

Exemplary results of experimental validation studies for each target areprovided in the Figures and are set forth below:

GFRa1

12 GFRa1 peptides were observed by mass-spec as over expressed in breastand kidney cancer cell lines, as follows: 19.2 to 127.3 foldover-expressed in breast cancer cell line and conditioned media, and 2.5to 14 fold over-expressed in kidney cancer cell line.

Immunohistochemistry (IHC) confirms expression of GFRa1 in breast (9%)and kidney (10%) tumors (FIG. 1).

IHC analysis indicates that GFRa1 staining of breast cancer samples doesnot correlate with ER, PR or HER2 status (FIG. 3).

Overexpression of GFRa1 was observed, both by FACS and by Taqman, inbreast cancer cell line MCF-7 and kidney cell line ACHN (FIG. 4). Thiscorrelates with over-expression of GFRa1 as observed by mass spec.

Elevated expression of GFRa1 mRNA was observed in breast tumors byTaqMan (FIGS. 5 and 18), correlating with elevated protein expressionobserved my mass-spec and IHC. GFRa1 kinase binding partner (Ret) mRNAand GFRa1 ligand (GDNF) mRNA were also over-expressed in breast tumors(FIGS. 19-20).

GFRa1 ligand (GDNF) is expressed in MCF-7 breast cancer cells (FIG. 17).Both GFRa1 and it's kinase binding partner (Ret) are expressed in MCF7and HCC1937 breast cancer cells (FIG. 21). GFRa1 is also expressed inACHN and Caki 1 kidney cancer cell lines (FIG. 24), and in breast cancercell lines and tumors (FIG. 26).

Functional data indicates that GFRa1 siRNA inhibits proliferation (35%)and induces apoptosis (3.4 fold) of Caki-1 kidney cancer cell lines(FIGS. 6-8), and also inhibits proliferation in lung cancer cells (FIG.6).

Functional data indicates that recombinant GFRa1 ligand (GDNF) increasesproliferation of MCF-7 breast cancer cells (FIGS. 9, 22-23, and 33).However, heat-denatured GFRa1 ligand does not induce MCF-7 cellproliferation (FIG. 28).

A monoclonal antibody to GFRa1 ligand (GDNF) blocks binding of GFRa1ligand (FIG. 30). Furthermore, GFRa1 ligand-mediated MCF-7 cellproliferation is blocked by neutralizing anti-GFRa1 ligand antibodies(FIG. 31).

Effect of GFRa1 ligand/GFRa1 antagonists on MCF-7 breast cancer cellproliferation in complete growth medium (no exogenous GFRa1 ligand) isshown in FIG. 32.

Inhibitors (e.g., small molecule kinase inhibitor compounds) of GFRa1kinase binding partner (Ret) inhibit proliferation induced by GFRa1ligand (GDNF) in MCF-7 breast cancer cells (FIG. 10).

GFRa1 peptide blocks 20 ng/ml GDNF (GFRa1 ligand) mediated MCF-7 breastcancer cell proliferation (FIG. 25).

GFRa1 kinase binding partner (Ret) but not GFRa1 is expressed in ASPC-1and BXPC-3 pancreatic cancer cells (FIG. 27).

IHC and Taqman indicated limited normal tissue expression of GFRa1.Elevated expression of GFRa1 was observed in the following normal cellsand tissues: brain, ganglion cells, and lymphocytes.

Claudin-4

A Claudin-4 peptide was observed by mass spec as over-expressed inbreast and gastric cancer cell lines (11.8-fold over-expressed in breastcancer cell line and 33.2-fold over-expressed in gastric cancer cellline).

Immunohistochemistry (IHC) indicates that Claudin-4 is over-expressed inmultiple tumor types, as follows: breast (over-expressed in 50% oftumors), ovarian (40%), and lung (20%) (FIG. 35). Specifically,Claudin-4 was over-expressed in 5 out of 10 breast cancer specimens, 4out of 10 ovarian cancer specimens, and 2 out of 10 lung cancerspecimens, as indicated by IHC.

Claudin-4 is localized mostly at the membrane of tumor epithelial cells,as indicated by IHC.

ASCT2

Five ASCT2 peptides were observed by mass spec as over-expressed incolon and gastric tumor tissues as well as in pancreatic, colon, lung,breast, liver, melanoma and gastric cancer cell lines, as follows:2.2-20.2-fold over-expressed in breast cancer cell lines, 5.1-6.0 foldin colon cancer cell line, 4.5-13.0 fold in colon cancer tissues, 2.8fold in kidney cancer endothelial cells, 3.1-6.6 fold in liver cancercell lines, 8.0-22.7 fold in lung cancer cell lines, 4.0-9.0 fold inpancreatic cancer cell line, 3.7-18.3 fold in melanoma cell lines,14.0-28.7 fold in gastric cancer cell lines, and 27.3-44.2 fold ingastric cancer tissue. ASCT2 was also over-expressed in renalendothelial cells.

IHC confirmed expression of ASCT2 in colon, lung, pancreatic, liver andgastric tumors. ASCT2 was over-expression in multiple tumor types, asindicated by IHC, as follows: metastatic pancreas (43%), prostate (40%),ovary (30%), and pancreas (22%) (FIG. 36).

For IHC, a commercially available rabbit polyclonal antibody raisedagainst an amino terminal peptide and an internally generated rabbitpolyclonal antibody raised against a peptide (amino acids 515-530 ofASCT2) were used.

mRNA expression analysis indicates over-expression of ASCT2 inpancreatic tumors (FIG. 37).

Knockdown of ASCT2 mRNA inhibited proliferation of pancreatic (40%), andcolon cancer cells, as well as breast cancer cells (FIGS. 38-40).

CD166-ALCAM

15 CD166 peptides were observed by mass-spec as over expressed in lung,colon and AML tissues and breast, gastric, kidney, lung, prostate andpancreatic cancer cell lines, as follows: 2.4 to 145.4 foldover-expressed in breast cancer cell lines and conditioned media, 3.5 to6 fold in colon tissues, 3.6 to 26.4 fold in gastric cell line, 4.6 foldin kidney cancer cell line, 3.2 to 61.7 fold in lung cancer tissues celllines and conditioned medium from lung cancer cell line, 4.4 to 27.2fold in prostate cancer cell lines and conditioned media, 5 to 19.8 foldin pancreatic cancer cell lines, and 3.2 to 7.3 fold in AML primarycells.

IHC confirms expression of CD166 in breast and colon tumors. IHCindicates that CD166 is over-expressed by 2 pathology grades in multipletumor specimens, as follows: breast (40%), bladder (30%), and colon(20%) (FIG. 42).

Fluorescence-activated cell sorting (FACS) indicates over-expression ofCD166 in multiple cell lines (breast, colon, and pancreas) and tumortissues (lung and colon) (FIGS. 43-44).

Elevated CD166 mRNA expression was observed in breast and colon tumortissues, and in pancreatic cell lines (FIGS. 45-47).

Functional data indicates that CD166 siRNA inhibits proliferation ofASPC-1 pancreatic cancer cell lines (33%), HT29 and HCT116 colon cancercell lines (35 to 64%), MCF-7 breast cancer cell lines (36%), Caki-1kidney cancer cell lines (59%), and AGS and NCI-N87 gastric cancer celllines (48 to 61%) (FIGS. 48, 50-53).

Functional data indicates that CD166 siRNA induces apoptosis of HCT116and HT29 colon cancer cell lines (1.5 to 2.8 fold) and AGS gastriccancer cell lines (2 fold) (FIGS. 49-50, 52).

CD166 siRNA in combination with Gemzar can enhance apoptosis of BXPC-3pancreatic cancer cells (FIG. 54).

Saporin-conjugated 2nd Ab+CD166 mAb induces cell death in CD166 positiveHCC1954 breast cells, but does not induce cell death in CD166 negativeHCC1937 breast cells (FIGS. 55-60).

High expression of CD166 was observed in the following normal cells andtissues: Adrenal Medulla, Liver Bile duct epithelium and peripheralnerves, Lung Airway, Ovary Follicle, Pancreas Islet of Langerhans,Prostate, Small Intestine Ganglion cell and Peripheral Nerves, ThymusEpithelium, and Uterus Endometrium

CD55

10 CD55 peptides were observed by mass spec as over-expressed in 22cancer cell lines and 15 tumor tissues, as follows: 2.4-14.5 foldover-expressed in breast cancer cell lines, 4.6-100 fold in colontissues, 3.0-19.9 fold in kidney cancer cell lines, 4.8-6.3 fold inliver cancer cell lines, 4.3-111.7 fold in lung cancer cell lines, 6.3fold in lung cancer cell line conditioned medium, 2.4-10 fold in lungtissues, 100 fold in pancreatic cancer cell line conditioned medium, 7fold in prostate cell line, 4.9 fold in melanoma cell line, and 9.3-49.7fold in gastric cell lines.

IHC indicates over expression of CD55 in multiple tumors types, asfollows: colon (30%), lung (squamous) (30%), melanoma, lymph node (30%),bladder (20%), pancreas (20%), and lung (adenocarcinoma) (10%) (FIGS. 62and 81).

Over-expression of CD55 mRNA was observed in colon and pancreatic tumortissue (FIGS. 66 and 73-78).

QFACS confirms over-expression of CD55 in 100% of colon tumors relativeto normal colon using a commercially available monoclonal antibody(FIGS. 63-65).

Knockdown of CD55 mRNA inhibits proliferation in multiple cell lines(35%), including colon and prostate cancer cell lines (FIG. 67).Functional data also indicate that CD55 siRNA inhibits proliferation ofHCT116 colon cancer cell line (90%) (FIG. 68).

TG2

11 TG2 peptides were observed by mass spec as over-expressed in lung,colon and kidney tumors; lung, colon, kidney, breast, prostate andpancreatic cancer cell lines and lung conditioned media, as follows:2-21 fold over-expressed in lung tumor samples, 3-19 fold in lung cancercell lines, 5 fold in lung conditioned media, 4-5 fold in colon tumorsamples, 10-12 fold in a colon cancer cell line, 3-6 fold in pancreaticcancer cell lines, 16 fold in a breast cancer cell line, 3-15 fold inkidney cancer cell lines, 7-15 fold in kidney tumor samples, and 4 foldin a prostate cancer cell line.

TG2 was over-expressed, as indicated by IHC, in multiple tumor types, asfollows: metastatic pancreatic tumors (100%), liver (50%), pancreas(44%), lung, NSC (20%), ovarian (20%), pharyngeal (20%), prostate (10%),gastric (10%), and esophageal (10%) (FIG. 83). TG2 showed limitedexpression in normal tissues, as measured by IHC and TaqMan.

Knockdown of TG2 mRNA inhibits proliferation in MPANC96 and BXPC-3pancreatic (29 and 49%), Calu-1 lung (35%), and HT29 colon cancer celllines (83%) (FIGS. 85-88).

TG2 mRNA over-expression was observed in pancreatic and colorectal tumortissue.

TG2 was over-expressed in skin wound healing granulation tissue, asindicated by IHC.

CRA032197 (a specific small molecule inhibitor of TG2) blocked A549 lungtumor cell line and HUVEC proliferation.

CRA31033 and CRA032197 inhibited A549 xenograft tumor growth in a dosedependent fashion.

CD49f

13 CD49f peptides observed by mass spec as over-expressed in colon,kidney and gastric tumors and prostate, lung, colon, breast, kidney,gastric, liver and melanoma cancer cell lines, as follows: 3-48 foldover-expressed in colon tumors, 4-10 fold in colon cell lines, 4-21 foldin lung cell lines, 2-16 fold in breast cell lines, 9 fold in a kidneycell line, 3-4 fold in kidney tumors, 9 fold in a melanoma cell line,6-14 fold in liver cell lines, 3-26 fold in prostate cell lines, 3-43fold in gastric cell lines, and 8-23 fold in gastric tumors.

CD49f is over-expressed in multiple tumor types, as indicated by IHC, asfollows: Kidney (100%), Lung, NSC (80%), Lung, Squamous (70%), Pancreas,Metastatic (67%), Melanoma, Lymph node (50%), Skin, Melanoma (50%),Brain, Glioblastoma (50%), Gastric (40%), Liver (38%), Pancreas (29%),Ovarian (20%), and Colon (20%) (FIG. 90).

CD49f is over-expressed in colon tumors (FIGS. 91-92).

Functional data indicates that CD49f siRNA inhibits proliferation of twolung cell lines (35% and 47%), two colon cell lines (32% and 68%), and agastric cell line (46%) (FIGS. 93-96).

Anti-CD49f antibody blocked H1299 lung tumor cell line proliferation(FIG. 97).

QFACS data indicate over-expression of CD49f in colon tumor epithelial.

CD49f is Over-expressed in Melanoma Cell Lines as Measured by FACS.

CD98

7 CD98 peptides were observed by mass spec as over-expressed in lung andcolon tumor tissues and lung, kidney, gastric, breast, liver, melanoma,esophageal and pancreatic cancer cell lines, as follows: 4.5fold-singleton over-expressed in colon tumor tissues, 2.9-239.5 foldover-expressed in lung tumor tissues and cancer cell lines, 3.6-10.2fold in kidney cancer cell lines, 10.9-13.6 fold in stomach cancer celllines, 3.2-7 fold in breast cancer cell lines, 3.5-11.3 fold in livercancer cell lines, 5.2-6.3 in melanoma cell lines, 145.6 fold inesophageal cancer cell line, and 5.2 fold in pancreatic cancer cellline.

IHC indicates over-expression of CD98 in multiple tumor types, asfollows: Lung adeno (100%), Lung squamous (100%), Melanoma (90%),Glioblastoma (83%), Colon (80%), and Breast (50%) (FIG. 99).

QFACS confirms over-expression of CD98 in lung and colon tumors (FIGS.101 and 104), which is consistent with results from mass spec and IHC.

Knockdown of CD98 inhibits proliferation in lung cancer cells, as wellas pancreas and breast cancer cells (FIGS. 106-109).

CD104

14 CD104 peptides were observed by mass spec as over-expressed in tumortissues (colon, lung, kidney, and gastric) and cancer cell lines(pancreatic, colon, lung, breast, kidney and gastric), as follows: 46-67fold over-expressed in 1 pancreatic cell line, 2-42 fold in multiplecolon tissues, 3-5 fold in 1 colon cell line, and as a singleton in 1colon cell line, 2-4 fold in 1 breast cell line, 3 fold in 1 lungtissue, 3-23 fold in 3 lung cell lines, 3-4 fold in 1 kidney tissue, 4-8fold in 2 kidney cell lines, 4-32 fold in 1 gastric tissue, and 6-38fold in nonmetastatic gastric cell lines compared to metastatic gastriccell lines.

IHC confirms over-expression of CD104 in multiple tumor types, asfollows: pancreas (40%) and colon (10%), and by 2 pathology grades inpancreas (30%) and colon (10%) (FIG. 111). IHC also indicatedover-expression of CD104 in breast, liver, pancreas, and gastric tumors(FIG. 112). The antibody used for IHC analysis of CD104 was a mousemonoclonal antibody raised against the cytoplasmic domain of CD 104.

Over-expression of CD104 mRNA was observed in pancreatic and colon tumortissues, as indicated by Taqman analysis (FIG. 114).

Knockdown of CD 104 mRNA inhibits proliferation in colon and breastcancer cell lines (FIGS. 115-116).

Over-expression of CD104 in colon tumors, as indicated by QFACS (FIG.113), confirms mass spec and IHC results.

DPEP1

Four DPEP1 peptides were observed by mass spec as over-expressed in 40%of colon tumor tissues (2.7-100 fold overexpressed in colon tissues).

IHC indicates over-expression of DPEP1 in 20% of colon tumors (FIG.118). DPEP1 was expressed at an intensity level of two in surfaceepithelium of normal colon, as indicated by IHC.

FACS confirms over-expression of DPEP1 on 45% of colon tumor tissuesrelative to normal colon using an internally generated monoclonalantibody (FIG. 119).

Over-expression of DPEP1 mRNA was observed in colorectal tumor tissue(FIG. 120).

Knockdown of DPEP1 mRNA inhibits proliferation of colon cancer celllines (72% and 56%) (FIGS. 121, 123-124) and induces apoptosis of acolon cancer cell line (2.2 fold) (FIGS. 122-123).

A monoclonal antibody to DPEP1 inhibits proliferation in colon cancercells (FIG. 125).

Tissue Factor (TF)

3 TF peptides were observed by mass spec as over-expressed inpancreatic, lung and breast cancer tumor cell lines, as follows: 3.5-7.6fold over-expressed in breast cancer cell lines, 10.3 fold inconditioned medium from breast cancer cell line, 2.5-4.2 fold in lungcancer cell lines, and 5.1-75.0 fold over-expressed in pancreatic cancercell lines.

TF is overexpressed by 2 pathology grades in multiple tumor types, asfollows (as indicated by IHC): pancreatic cancer (72%), metastaticpancreatic tumors (67%), liver cancer (40%), prostate cancer (20%) andcolorectal adenocarcinomas (10%) (FIG. 128).

Cell surface overexpression of TF was confirmed by FACS in pancreaticand colorectal cell lines and lung tumor specimens (FIG. 137).

mRNA profiling indicated over-expression of TF mRNA in pancreatic andlung tumor tissues (FIGS. 131 and 136).

Knockdown of TF mRNA inhibits proliferation in pancreatic, lung, colon,gastric and prostate cell lines (FIGS. 132-134).

TF ligand activates the AKT signaling pathway (FIG. 135).

TF demonstrated limited expression in normal tissues, as indicated byIHC and Taqman.

QFACS indicated TF cell surface copy number >2.5×105 in pancreatic celllines.

Na—K ATPase Beta3

Six Na—K ATPase β3 peptides were observed by mass-spec as over-expressedin multiple lung, colon and kidney tissues and lung, colon andpancreatic cancer cell lines, as follows: 3.4 to 23.5 foldover-expressed in lung cancer cell lines and tissues, 6.7 to 9.3 fold incolon cell line and tissue, 4.2 to 9.1 fold in kidney tissue, and 100fold (singleton) in pancreatic cell line.

IHC confirms expression of Na—K ATPase β3 in lung and pancreatic tumors.

IHC indicates overexpression of Na—K ATPase β3 by 2 pathology grades inmultiple tumor specimens, as follows: Brain (83%), Lung, NSC (80%),Pancreas (71%), Breast (70%), Melanoma (70%), Melanoma, Lymph Node(70%), Metastatic Pancreatics (67%), Lung, Squamous (60%), Colon (50%),Ovary (50%), Liver (38%), and Gastric (30%) (FIG. 139).

Taqman indicates overexpression of Na—K ATPase β3 mRNA in multiplepancreatic tumors and several pancreatic cancer cell lines.

Functional data indicates that Na—K ATPase β3 siRNA inhibitsproliferation of ASPC-1 and MPANC96 pancreatic cell lines (51 to 56%),Calu-1 lung cell lines (28%), HCT116 colon cell line (84%), and Caki-1kidney cell lines (52%) (FIGS. 141 and 143-145).

Functional data indicates that Na—K ATPase β3 siRNA induces apoptosis ofASPC-1 and MPANC96 pancreatic cell lines (1.9 to 2.9 fold), Calu-1 lungcell lines (2.2 fold), MCF-7 breast cell lines (1.7 fold), Caki-1 kidneycell lines (5.8 fold), and AGS and NCI-N87 gastric cell lines (1.6 fold)(FIGS. 142-145).

Anti-Na—K ATPase β3 antibody inhibits cell proliferation in ASPC-1 andBXPC-3 pancreatic cancer cells (FIG. 146).

Na—K ATPase β3 siRNA in combination with Gemzar increases apoptosis ofBXPC-3 pancreatic cancer cells (FIG. 147).

High expression of Na—K ATPase β3 was observed in the following normalcells and tissues: adrenal medulla, bone marrow erythroid and myeloidprecursors, PBLs, platelets, epithelium of the esophagus, kidney,testis, pharynx, and prostate.

VIPR1

A VIPR1 peptide was observed by mass spec as over-expressed in breastand melanoma cancer cell lines (2.3-16 fold over-expressed in breastcancer cell lines and 6 fold over-expressed in melanoma cancer cellline).

IHC indicates over-expression of VIPR1 in multiple tumors types, asfollows: Non-Hodgkin's Lymphoma, Lymph Nodes (100%), Bladder (50%), Lung(Squamous) (40%), Ovary (40%), Liver (38%), Metastatic Pancreatic (33%),Esophageal (20%), and Pharyngeal (20%) (FIG. 149).

Knockdown of VIPR1 mRNA inhibits cell proliferation in lung, colon,breast and gastric cell lines (FIGS. 151-154).

Antibody to VIPR1 inhibits proliferation in colon and breast cancercells (FIG. 155).

CD26

12 CD26 peptides were observed by mass spec as over-expressed in colon,kidney, lung, and gastric tumor tissues and cell lines, and in liver andprostate cell lines, as follows: 13.8 fold over-expressed in colon cellline, 2.3-100 fold in colon tissues, 3.8-43.92 fold in kidney celllines, 3.3 fold in kidney tissue, 13.4-18.6 fold in liver cell lines, 8fold in lung cell line, 5-30.2 fold in lung tissues, 4.9-11.2 fold inprostate cell lines, 4.7-20 fold in gastric cell lines, and 36.2 fold ingastric tissue.

IHC indicates over-expression of CD26 in multiple tumor types, asfollows: colon (50%), prostate (30%), non-Hodgkin's lymphoma (17%), andkidney (10%) (FIG. 157).

FACS indicates that CD26 is over-expressed in colon and lung tumortissues relative to normal tissue (FIGS. 159-160).

Knockdown of CD26 mRNA inhibits cell proliferation in lung (31% and50%), gastric (41 and 34%), colon (42%), breast (30%), and kidney (29%)cancer cells (FIGS. 162 and 164-167).

Knockdown of CD26 inhibits proliferation and induces apoptosis inH1299-HES spheroid cancer cells (cancer stem cells) (FIGS. 163 and 168).

Monoclonal antibody to CD26 inhibits cell proliferation in lung andcolon cancer cell lines (FIG. 169).

CXADR

4 CXADR peptides were observed by mass-spec as over-expressed in breast,kidney, and lung cancer cell lines and in colon tumor tissues, asfollows: 4-100 fold over-expressed in 2 breast cancer cell lines, 3-100fold in 6 colon tumor tissues, 6-14 fold in a kidney cancer cell line,and 7 fold in a lung cancer cell line.

IHC indicates over-expression of CXADR in multiple tumors, as follows:non-Hodgkin's lymphoma (67%), Ovarian (60%), Brain glioblastoma (17%),Liver (13%), and Colon (FIG. 171).

CXADR mRNA over-expression was observed in colon tumor tissue (FIGS. 174and 179).

Knockdown of CXADR mRNA mediates a decrease in proliferation in H1299and Calu-1 lung (25%), HCT116 and HT29 colon (70 and 45%), and NCI-N87gastric (39%) cancer cell lines (FIGS. 175-177).

Functional validation results support over expression of CXADR in colonand lung tumors as indicated by mass spec.

Anti-CXADR monoclonal antibody reduces proliferation in colon and lungcancer cell lines (FIG. 178).

CXADR expression was observed by FACS on colon and lung cancer celllines (FIG. 172) and breast tumor tissue. CXADR was also over-expressedin 3D spheroid cancer cells (cancer stem cells) derived from a kidneycancer cell line (FIG. 173).

Mass spec cross-tissue analysis of CXADR reveals elevated expression ofCXADR in breast, kidney, and colon tumors.

PTK7

13 PTK7 peptides were observed by mass-spec as over-expressed inprostate, kidney, lung, breast, and gastric cancer cell lines and ingastric cancer tissue and breast conditioned media (CM), as follows: 38fold over-expressed in a prostate cell line, 17 fold in a kidney cellline, 5-70 fold in lung cancer cell lines, 3-15 fold in breast cancercell lines, 7-32 in gastric cell lines, 5 fold in a gastric tissue, and6-15 fold over-expressed in breast CM.

PTK7 is over-expressed in multiple tumor types, as indicated by IHC, asfollows: Prostate (70%), Brain, Glioblastoma (67%), and Kidney (50%), aswell as Colon (10%) and Bladder (10%) (FIG. 183). IHC also indicatedover-expression of PTK7 in lung tumor tissues (PTK7 was expressed in 7out of 10 lung tumor specimens).

Over-expression of PTK7 mRNA was observed (by TaqMan) in multiple cancercell lines and tissues including prostate, lung, and colon (FIG. 187).

Knockdown of PTK7 mRNA inhibits proliferation of the following cancercell lines: Calu1 Lung cell line (31%), H1299 lung cell line (58%),HCT116 colon cell line (71%), MPANC96 pancreatic cell line (39%), ACHNkidney cell line (37%) and NCI-N87 gastric cell line (48%) (FIGS. 188,190-191)).

Knockdown of PTK7 mRNA induces apoptosis of the following cancer cellslines: Calu1 Lung cell line (85%), H1299 lung cell line (865%), ASPC-1pancreatic cell line (50%), Caki-1 kidney cell line (68%), and prostate(2.4 fold) (FIGS. 189-190).

A rabbit polyclonal antibody to PTK7 blocked cell proliferation in H1299lung tumor cell line (FIG. 192).

PTK7 is expressed in a hormone-refractory prostate xenograft model(FIGS. 184 and 195).

PTK7 expression was observed on 3D tumor spheroid cells (cancer stemcells) (FIGS. 186, 194, and 203).

PTK7 is expressed on prostate cancer cell lines, as measured by flowcytometry (FIGS. 196-201).

PTK7 mRNA expression in prostate tumors and normal tissues demonstratestwo populations in prostate tumors (FIG. 202).

PTK7 is expressed in the following normal tissues: cardiac myocytes,pancreatic islets, hepatocytes, and bone marrow precursors.

MISTR

Two MISTR peptides were observed by mass spec as over-expressed inbreast and prostate cancer cell lines, as follows: 3.3-8.8 foldover-expressed in breast cancer cell lines and 2.0-4.9 foldover-expressed in prostate cancer cell lines.

IHC indicates over-expression of MISTR in multiple tumors types, asfollows: Ovary (90%), Liver (75%), Colon (70%), Lung (NSC), (70%),Breast (50%), Pancreas (38%), and Metastatic Pancreatic (100%). IHC alsoindicated over-expression of PTK7 in lung tumor tissues (PTK7 wasexpressed in 7 out of 10 lung tumor specimens) (FIG. 206).

Over-expression of MISTR mRNA was observed in pancreatic, colon, andovarian tumor tissue (FIG. 207).

Knockdown of MISTR mRNA inhibits proliferation in pancreatic (38-54%),lung (28-39%), colon (72-95%), kidney (59%) and gastric cell lines (47%)(FIGS. 208 and 210-211).

Knockdown of MISTR mRNA induces apoptosis in pancreatic (2 fold), lung(1.4 fold), and colon (1.5-2.1 fold) cell lines (FIG. 209).

An antibody to MISTR inhibits proliferation in lung cancer cells (FIG.212).

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention, which are obvious to those skilled inthe field of molecular biology or related fields, are intended to bewithin the scope of the following claims.

The invention claimed is:
 1. A method for identifying a human as havingovarian cancer, the method comprising detecting the level of Na—K ATPasebeta3 protein in a sample from the human, and identifying said human ashaving ovarian cancer if the level is elevated relative to a controlNa—K ATPase beta3 protein level established for a non-cancerous sample.2. The method of claim 1, wherein the amino acid sequence of the Na—KATPase beta3 protein comprises a sequence selected from the groupconsisting of SEQ ID NOS:127-134.
 3. The method of claim 1, wherein theamino acid sequence of the Na—K ATPase beta3 protein consists of asequence selected from the group consisting of SEQ ID NOS:127-134. 4.The method of claim 1, wherein the level of Na—K ATPase beta3 protein isdetected by contacting the sample with an antibody that selectivelybinds to the Na—K ATPase beta3 protein, and detecting binding of theantibody to the Na—K ATPase beta3 protein.
 5. The method of claim 4,wherein the antibody is coupled to a detectable substance.
 6. The methodof claim 5, wherein the detectable substance comprises a fluorescentlabel.
 7. The method of claim 1, wherein the level of Na—K ATPase beta3protein is detected by immunohistochemistry (IHC).
 8. The method ofclaim 1, wherein the sample comprises ovarian tissue.